Enhanced antisense oligonucleotides

ABSTRACT

Described herein are gap-widened antisense oligonucleotides having improved therapeutic index as compared to 5-10-5 MOE gapmer antisense oligonucleotides of the same sequence. Also described are methods of reducing a target RNA in an animal using the gap-widened antisense oligonucleotides of the present invention. Further, are methods for selecting a gap-widened antisense oligonucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/231,243, filed Sep. 19, 2005, allowed Oct. 19, 2010, which claimspriority under 35 USC 119(e) to U.S. Application No. 60/611,100, filedon Sep. 17, 2004 and to U.S. Application No. 60/663,442, filed on Mar.18, 2005, each of which is herein incorporated by reference in itsentirety. The instant application is also related to U.S. Application60/718,685, filed Sep. 19, 2005, and U.S. Application 60/718,684, filedSep. 19, 2005, each of which is herein incorporated by reference in itsentirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0051USC1SEQ.txt, created on Jan. 17, 2011 which is 92 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides chimeric antisense compounds havingenhanced in vivo potency and thus an improved therapeutic index. Thecompounds described herein have widened deoxy gaps and enhanced in vivopotency which is unexpected based on their in vitro activity.

BACKGROUND OF THE INVENTION

Antisense oligonucleotides are accepted therapeutic modalities and manythousands of patients have been treated with antisense compounds. Theoriginal “first generation” antisense compounds employed in the firstantisense clinical trials were oligodeoxynucleotides having 2′-deoxyribonucleotides and phosphorothioate intemucleoside linkages.Subsequently, chimeric “second generation” antisense oligonucleotidesexhibited a marked improvement in potency over first generationantisense oligonucleotides. Second generation antisense oligonucleotidesare chimeric oligonucleotides typically having a 2′-deoxy “gap” regionflanked by “wings” having nucleotides with 2′-modified ribonucleotides,referred to as “gapmers.” The most widely used of the “secondgeneration” antisense motifs is often referred to as a “MOE gapmer” inwhich the 2′-modified ribonucleotide is a 2′-O-methoxyethyl (2′-MOE orsimply MOE) modification, and each of the internucleotide linkages is aphosphorothioate. Predominantly, second generation oligonucleotides havea length of 20 nucleotides of which the 5 nucleotides at each terminusare 2′-MOE nucleotides and the center ten nucleotides are2′-deoxyribonucleotides. These second generation oligonucleotides arereferred to as “5-10-5 MOE gapmers” have a 5-10-5 wing-gap-wing motif.Chimeric antisense compounds with other arrangements of modificationshave also been made. “Hemimers,” are chimeric compounds in which thereis a single 2′-modified “wing” adjacent to (on either the 5′, or the 3′side of) a 2′-deoxy gap have been described (Geary et al., 2001, J.Pharm. Exp. Therap., 296, 898-904).

SUMMARY OF THE INVENTION

The present invention is directed to “gap-widened” antisenseoligonucleotides having a gap region of greater than 112′deoxyribonucleotides flanked by two “wing” regions having from one toeight nucleotides which do not support RNase H activity. The gap-widenedantisense oligonucleotide of the present invention have been shown tohave an improved therapeutic index as compared to a correspondingantisense oligonucleotide having a 5-10-5 MOE gamer antisenseoligonucletide with the same sequence. The gap-widened antisenseoligonucleotides of the present invention exhibit increased in vivopotency or improved tissue exposure as compared with the corresponding5-10-5 MOE gapmer antisense oligonucleotide with the same sequence. Mostinterestingly, there is a lack of correlation between the in vitropotency and the in vivo potency of the gap-widened antisenseoligonucleotides described herein. The gap-widened antisenseoligonucleotides of the present invention are 18 to 24 nucleotides inlength. In particular, the gap-widened antisense oligonucleotides of thepresent invention have wing regions having2′-O-(2-methoxyethyl)ribonucleotides.

In an additional embodiment of the present invention is a method ofreducing expression of a target RNA in an animal in need of reducingexpression of said target RNA, comprising administering to said animal agap-widened antisense oligonucleotide 18 to 24 nucleotides in lengthcomprising: a gap region having greater than 11 contiguous2′-deoxyribonucleotides; and a first wing region and a second wingregion flanking the gap region, wherein each of said first and secondwing regions independently have 1 to 82′-O-(2-methoxyethyl)ribonucleotides, having an improved therapeuticindex as compared to a corresponding 5-10-5 MOE gapmer antisenseoligonucleotide having a gap region of 10 contiguous2′-deoxyribonucleotides and a first wing region and a second wing regionflanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides. Theimprovement in therapeutic index is characterized by equal or increasedpotency coupled with a reduction in tissue concentration, or increasedpotency coupled with equal tissue exposures as compared to acorresponding 5-10-5 MOE gapmer antisense oligonucleotide of the samesequence. In addition, the improvement in therapeutic index may becharacterized by an increased liver to kidney concentration ratio ascompared to a corresponding 5-10-5 MOE gapmer antisense oligonucleotideof the same sequence. In particular, the method of the present inventionis useful in reducing the expression of RNA targets expressed in thekidney, liver, or adipose tissues. The method of the present inventionis also useful in reducing the expression of target RNA associated witha metabolic or cardiovascular disease or condition. The method of thepresent invention is useful wherein the metabolic disease or conditionis selected from diabetes, hepatic steatosis, fatty liver disease,non-alcoholic steatohepatitis, metabolic syndrome, obesity, or the like.In addition, the method of the present invention is useful wherein thecardiovascular disease or condition is selected fromhypercholesterolemia, atherosclerosis, hyperlipidemia, familialhypercholesterolemia, or the like.

An additional method of the present invention is a method of selecting agap-widened antisense oligonucleotide with an improved therapeuticindex, the method comprising:

screening in vitro a plurality of antisense oligonucleotides targeting ahuman RNA and having a single wing-gap-wing motif;

identifying a parent antisense oligonucleotide from the plurality ofantisense oligonucleotides having a potent in vitro activity;

synthesizing a plurality of gap-widened antisense oligonucleotideshaving the same sequence as the parent antisense oligonucleotide,wherein said gap-widened antisense oligonucleotide is 18 to 24nucleotides in length comprising a gap region having greater than 11contiguous 2′-deoxyribonucleotides; and a first wing region and a secondwing region flanking the gap region, wherein each of said first andsecond wing regions independently has 1 to 82′-O-(2-methoxyethyl)ribonucleotides;

testing said plurality of gap-widened antisense oligonucleotides in aplurality of animals;

obtaining potency and tissue concentration data from said testing step;and

determining an optimized gap-widened oligonucleotide wing-gap-wing motifwith an improved therapeutic index, improved potency, reduced tissueexposure, or reduced toxicity, or a combination thereof as compared tothe parent antisense oligonucleotide.

In one embodiment, the method of selecting a gap-widened antisenseoligonucleotide further comprises the step of designing a rodentsequence analogous or a non-human primate sequence to said parentantisense oligonucleotide. In one embodiment, the step of determiningthe optimized gap-widened antisense oligonucleotide wing-gap-wing motifwith an improved therapeutic index includes identifying a gap-widenedantisense oligonucleotide which has equal or increased potency ascompared to the parent antisense oligonucleotide.

In the step of screening, each of said antisense oligonucleotides hasthe same wing-gap-wing motif selected from 2-16-2, 3-14-3, 4-12-4, or5-10-5. In a further embodiment, the wing portions of the gap-widenedantisense oligonucleotides are 2′-O-(2-methoxyethyl)ribonucleotides. Inparticular, the step of screening is performed in primary hepatocytes,HepG2, bEND, or HeLa cells. In the step of identifying, the potent invitro activity is greater than 50% reduction in the target mRNAexpression as compared to a saline control. In alternate embodiments, inthe step of identifying, the potent in vitro activity is greater than30%, greater than 40%, greater than 50%, greater than 60%, greater than70%, greater than 80%, or greater than 90%

In the step of synthesizing, the gap-widened antisense oligonucleotideseach have different wing-gap-wing motifs. In particular, the gap-widenedantisense oligonucleotides have gaps of 12, 13, 14, 15, 16, 17, or 182′-deoxyribonucleotides in length. In the step of testing, the animalsare selected from rodents such as mice and rats, and non-human primates,such as cynomolgous monkeys.

In the step of obtaining, the tissue concentration data areconcentrations of full-length gap-widened antisense oligonucleotidesparticularly measured in the liver, kidney, or adipose tissue. In oneembodiment, each optimized gap-widened antisense oligonucleotide isselected because of equal or improved potency data. In anotherembodiment, each optimized gap-widened antisense oligonucleotide isselected because of reduced tissue exposure. In another embodiment, eachoptimized gap-widened antisense oligonucleotide is selected because ofreduced toxicity. In another embodiment, each optimized gap-widenedantisense oligonucleotide is selected because of improved therapeuticindex. In another embodiment, each optimized gap-widened antisenseoligonucleotide is selected because of reduced tissue exposure, reducedtoxicity, improved potency, or a combination thereof.

The gap-widened antisense oligonucleotides described herein may havevarious wing-gap-wing motifs selected from: 1-16-1, 2-15-1, 1-15-2,1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1,2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3,1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1, 2-14-3,3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5,5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3,4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1,2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1,2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5,5-12-3, 4-12-4, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5,5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1,2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5,5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3,4-12-4, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4,1-19-1, 1-18-2, 2-18-1, 1-17-3, 3-17-1, 2-17-2, 1-16-4, 4-16-1, 2-16-3,3-16-2, 1-15-5, 2-15-4, 4-15-2, 3-15-3, 1-14-6, 6-14-1, 2-14-5, 5-14-2,3-14-4, 4-14-3, 1-13-7, 7-13-1, 2-13-6, 6-13-2, 3-13-5, 5-13-3, 4-13-4,1-12-8, 8-12-1, 2-12-7, 7-12-2, 3-12-6, 6-12-3, 4-12-5, 5-12-4, 2-11-8,8-11-2, 3-11-7, 7-11-3, 4-11-6, 6-11-4, 5-11-5, 1-20-1, 1-19-2, 2-19-1,1-18-3, 3-18-1, 2-18-2, 1-17-4, 4-17-1, 2-17-3, 3-17-2, 1-16-5, 2-16-4,4-16-2, 3-16-3, 1-15-6, 6-15-1, 2-15-5, 5-15-2, 3-15-4, 4-15-3, 1-14-7,7-14-1, 2-14-6, 6-14-2, 3-14-5, 5-14-3, 4-14-4, 1-13-8, 8-13-1, 2-13-7,7-13-2, 3-13-6, 6-13-3, 4-13-5, 5-13-4, 2-12-8, 8-12-2, 3-12-7, 7-12-3,4-12-6, 6-12-4, 5-12-5, 3-11-8, 8-11-3, 4-11-7, 7-11-4, 5-11-6, 6-11-5,1-21-1, 1-20-2, 2-20-1, 1-20-3, 3-19-1, 2-19-2, 1-18-4, 4-18-1, 2-18-3,3-18-2, 1-17-5, 2-17-4, 4-17-2, 3-17-3, 1-16-6, 6-16-1, 2-16-5, 5-16-2,3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6, 6-15-2, 3-15-5, 5-15-3, 4-15-4,1-14-8, 8-14-1, 2-14-7, 7-14-2, 3-14-6, 6-14-3, 4-14-5, 5-14-4, 2-13-8,8-13-2, 3-13-7, 7-13-3, 4-13-6, 6-13-4, 5-13-5, 1-12-10, 10-12-1,2-12-9, 9-12-2, 3-12-8, 8-12-3, 4-12-7, 7-12-4, 5-12-6, 6-12-5, 4-11-8,8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1,2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3,1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6,6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6,6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4,5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4,5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6. In aparticular embodiment, the gap-widened antisense oligonucleotides of thepresent invention have a 2-16-2, 3-14-3, or 4-12-4 wing-gap-wing motif.

Another aspect of the present invention is the use of a gap-widenedantisense oligonucleotide 18-24 nucleotides in length comprising: a gapregion having greater than 11 contiguous 2′-deoxyribonucleotides; and afirst wing region and a second wing region flanking the gap region,wherein each of said first and second wing regions independently have 1to 8 2′-O-(2-methoxyethyl)ribonucleotides, having an improvedtherapeutic index as compared to a corresponding 5-10-5 antisenseoligonucleotide having a gap region of 10 contiguous2′-deoxyribonucleotides and a first wing region and a second wing regionflanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides in themanufacture of a medicament for the treatment of disorders and diseasesrelated to target RNA levels. Another embodiment of the presentinvention is a pharmaceutical composition comprising a gap-widenedantisense oligonucleotide 18-24 nucleotides in length comprising: a gapregion having greater than 11 contiguous 2′-deoxyribonucleotides; and afirst wing region and a second wing region flanking the gap region,wherein each of said first and second wing regions independently have 1to 8 2′-O-(2-methoxyethyl)ribonucleotides, having an improvedtherapeutic index as compared to a corresponding 5-10-5 antisenseoligonucleotide having a gap region of 10 contiguous2′-deoxyribonucleotides and a first wing region and a second wing regionflanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides andoptionally a pharmaceutically acceptable carrier, diluent, enhancer orexcipient. Another embodiment of the present invention is a gap-widenedantisense oligonucleotide 18-24 nucleotides in length comprising: a gapregion having greater than 11 contiguous 2′-deoxyribonucleotides; and afirst wing region and a second wing region flanking the gap region,wherein each of said first and second wing regions independently have 1to 8 2′-O-(2-methoxyethyl)ribonucleotides, having lower kidneyaccumulation as compared to a corresponding 5-10-5 antisenseoligonucleotide having a gap region of 10 contiguous2′-deoxyribonucleotides and a first wing region and a second wing regionflanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides asmeasured by plasma protein binding capacity of said gap-widenedantisense oligonucleotide. Also provided is a method of modulating geneexpression in an animal comprising the step of contacting said animalwith the pharmaceutical composition. Another embodiment is a method ofmodulating gene expression in an animal comprising the step ofcontacting said animal with a gap-widened antisense oligonucleotide ofthe invention wherein the accumulation of the gap-widened antisenseoligonucleotide in the kidney is less compared to a corresponding 5-10-5antisense oligonucleotide having a gap region of 10 contiguous2′-deoxyribonucleotides and a first wing region and a second wing regionflanking the gap region of 5 2′-O-(2-methoxyethyl)ribonucleotides. Inone embodiment, the kidney accumulation is measured by plasma proteinbinding capacity of said gap-widened antisense oligonucleotide.

Another embodiment of the present invention is a method of reducinglevels of a preselected RNA target in the liver of an animal comprisingadministering to said animal a chimeric antisense compound 11 to 80nucleobases in length which is targeted to said preselected RNA targetwherein said chimeric antisense compound comprises a first gap regionconsisting of at least 10 contiguous 2′-deoxynucleotides and a wingregion which consists of from 1 to 4 contiguous nucleosides ornucleoside analogs which are not substrates for RNaseH. In particularembodiments, said first gap region consists of at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, or atleast 18 contiguous 2′-deoxynucleotides. In one embodiment, the chimericantisense compound comprises second wing region which consists of from 1to 7 contiguous nucleosides or nucleoside analogs which are notsubstrates for RNase H, and wherein said gap region is located betweensaid first wing region and said second wing region. In anotherembodiment, the chimeric antisense compound is a chimeric antisenseoligonucleotide, and the nucleosides or nucleoside analog which is not asubstrate for RNase H is a nucleotide having a 2′ modification of thesugar moiety. In one embodiment, the nucleotide having a 2′ modificationof the sugar moiety is a 2′-O-methoxyethyl nucleotide. In someembodiments the compound is a 2-16-2 MOE gapmer, a 3-12-3 MOE gapmer, a3-10-7 MOE gapmer or a 7-10-3 MOE gapmer. In one embodiment, thechimeric antisense oligonucleotide has at least one phosphorothioatebackbone linkage.

Another embodiment of the present invention is a pharmaceuticalcomposition for use in reducing levels of a preselected RNA target inthe liver of an animal comprising a chimeric antisense compound targetedto said preselected RNA target, wherein said chimeric antisense compoundcomprises a first gap region consisting of at least 10 contiguous2′-deoxynucleotides and a wing region which consists of from 1 to 4contiguous nucleosides or nucleoside analogs which are not substratesfor RNase H.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Depicts a comparison of in vivo effects of a 5-10-5 MOE gapmer(SEQ ID NO: 1) and the corresponding gap-widened 2-16-2 MOE gapmer (SEQID NO: 1).

FIG. 2. Depicts a comparison the persistence of target mRNA modulationof a 5-10-5 MOE gapmer (SEQ ID NO: 1) and the corresponding gap-widened2-16-2 MOE gapmer (SEQ ID NO: 1).

FIG. 3. Depicts a comparison of in vitro effects of a 5-10-5 MOE gapmerand the corresponding gap-widened 2-16-2 MOE gapmer.

FIG. 4. Depicts a comparison of the concentrations of a 5-10-5 MOEgapmer and the corresponding gap-widened 2-16-2 MOE gapmer in liver andkidney tissues.

FIG. 5. Depicts a comparison of the in vitro effects of oligonucleotideshaving the same sequence (SEQ ID NO:3) but varied wing-gap-wing motifs.

FIG. 6. Depicts a comparison of the in vivo effects of oligonucleotideshaving the same sequence (SEQ ID NO: 3) but varied wing-gap-wing motifs.

DETAILED DESCRIPTION OF THE INVENTION

Certain gap sizes are optimal for in vivo efficacy of antisensecompounds. Surprisingly, improved potency (3-10× improvement) in mouseor rat liver has been demonstrated for gap-widened antisenseoligonucleotides compared to standard 5-10-5 MOE gapmer (for example,2-16-2, 2-14-2, 3-12-3 gapmers) antisense oligonucleotides. This hasbeen shown for several distinct antisense targets and this improvedpotency is not observed in cultured cells transfected with the samegap-widened antisense oligonucleotides. Thus the “gap-widened” motifsappear to convey some benefit to in vivo potency, particularly in theliver. It is demonstrated herein that chimeric antisense compoundshaving a gap of greater than eleven contiguous deoxynucleotides flankedby wing regions consisting of from 1 to 4 nucleotides which are notsubstrates for RNase H are particularly effective at reducing target RNAlevels in vivo, particularly in the liver.

Therapeutic Index

Therapeutic index is a measure which relates the dose of a drug requiredto produce a specified effect to that which produces an undesiredeffect. In one embodiment, improved therapeutic index of a gap-widenedantisense oligonucleotide is characterized by equal or increased potencyand a reduction in tissue concentration. In another embodiment, improvedtherapeutic index of a gap-widened antisense oligonucleotide ischaracterized by increased potency and equal tissue concentrations ascompared to a corresponding 5-10-5 antisense oligonucleotide. In anotherembodiment, improved therapeutic index of a gap-widened antisenseoligonucleotide is characterized by increased potency and decreasedtoxicity as compared to a corresponding 5-10-5 antisenseoligonucleotide. In another embodiment, improved therapeutic index of agap-widened antisense oligonucleotide is characterized by comparablepotency and decreased toxicity as compared to a corresponding 5-10-5antisense oligonucleotide. In some embodiments, the toxicity is renaltoxicity. In some embodiments, the toxicity is hepatic toxicity.

Indications

An embodiment of the present invention is a method of treating a diseaseor condition wherein a target RNA is associated with said disease orcondition by administering a compound of the invention. Anotherembodiment of the present invention is a method of preventing ordelaying the onset of a disease or condition wherein a target RNA isassociated with said disease or condition by administering a compound ofthe invention. Diseases or conditions include metabolic andcardiovascular diseases or conditions. In some embodiments, the diseaseor condition is metabolic syndrome, diabetes, obesity, hyperlipidemia,hypercholesterolemia, hypertriglyceridemia, Type 2 diabetes,diet-induced obesity, hyperglycemia, or insulin resistance. In oneembodiment, the disease or condition is hepatic steatosis. In someembodiments, the steatosis is steatohepatitis or NASH. In someembodiments, the disease or condition is familial hypercholesterolemia,nonfamilial hypercholesterolemia, mixed dyslipidemia,dysbetalipoproteinemia, atherosclerosis, coronary artery disease,myocardial infarction, hypertension, carotid artery diseases, stroke,cerebrovascular disease, carotid artery disease, stroke, cerebrovasculardisease, peripheral vascular disease, thrombosis, or arterial aneurism.

NAFLD and Metabolic Syndrome

The term “nonalcoholic fatty liver disease” (NAFLD) encompasses adisease spectrum ranging from simple triglyceride accumulation inhepatocytes (hepatic steatosis) to hepatic steatosis with inflammation(steatohepatitis), fibrosis, and cirrhosis. Nonalcoholic steatohepatitis(NASH) occurs from progression of NAFLD beyond deposition oftriglycerides. A second-hit capable of inducing necrosis, inflammation,and fibrosis is required for development of NASH. Candidates for thesecond-hit can be grouped into broad categories: factors causing anincrease in oxidative stress and factors promoting expression ofproinflammatory cytokines. It has been suggested that increased livertriglycerides lead to increased oxidative stress in hepatocytes ofanimals and humans, indicating a potential cause-and-effect relationshipbetween hepatic triglyceride accumulation, oxidative stress, and theprogression of hepatic steatosis to NASH (Browning and Horton, J. Clin.Invest., 2004, 114, 147-152). Hypertriglyceridemia andhyperfattyacidemia can cause triglyceride accumulation in peripheraltissues (Shimamura et al., Biochem. Biophys. Res. Commun., 2004, 322,1080-1085).

“Metabolic syndrome” is defined as a clustering of lipid and non-lipidcardiovascular risk factors of metabolic origin. It is closely linked tothe generalized metabolic disorder known as insulin resistance. TheNational Cholesterol Education Program (NCEP) Adult Treatment Panel III(ATPIII) established criteria for diagnosis of metaolic syndrome whenthree or more of five risk determinants are present. The five riskdeterminants are abdominal obesity defined as waist circumference ofgreater than102 cm for men or greater than 88 cm for women, triglyceridelevels greater than or equal to 150 mg/dL, HDL cholesterol levels ofless than 40 mg/dL for men and less than 50 mg/dL for women, bloodpressure greater than or equal to 130/85 mm Hg and fasting glucoselevels greater than or equal to 110 mg/dL. These determinants can bereadily measured in clinical practice (JAMA, 2001, 285, 2486-2497).

HbA1c

HbA1c is a stable minor hemoglobin variant formed in vivo viaposttranslational modification by glucose, and it contains predominantlyglycated NH2-terminal β-chains. There is a strong correlation betweenlevels of HbA1c and the average blood glucose levels over the previous 3months. Thus HbA1c is often viewed as the “gold standard” for measuringsustained blood glucose control (Bunn, H. F. et al., 1978, Science. 200,21-7). HbA1c can be measured by ion-exchange HPLC or immunoassay; homeblood collection and mailing kits for HbA1c measurement are now widelyavailable. Serum fructosamine is another measure of stable glucosecontrol and can be measured by a colorimetric method (Cobas Integra,Roche Diagnostics).

Cardiovascular Risk Profile

Conditions associated with risk of developing a cardiovascular diseaseinclude, but are not limited to, history of myocardial infarction,unstable angina, stable angina, coronary artery procedures (angioplastyor bypass surgery), evidence of clinically significant myocardialischemia, noncoronary forms of atherosclerotic disease (peripheralarterial disease, abdominal aortic aneurysm, carotid artery disease),diabetes, cigarette smoking, hypertension, low HDL cholesterol, familyhistory of premature CHD, obesity, physical inactivity, elevatedtriglyceride, or metabolic syndrome (Jama, 2001, 285, 2486-2497; Grundyet al., Circulation, 2004, 110, 227-239).

EXAMPLES Example 1 Oligonucleotide Sequences and Targets

TABLE 1 Oligonucleotide sequences (all are PS backbone)Modified nucleotides are shown in Bold (2′MOE unless otherwiseindicated) and all cytosines are 5-methylcytosines SEQ ID ISIS No.Target Sequence NO Motif 116847 PTEN CTGCTAGCCTCTGGATTTGA 1 5-10-5344266 PTEN CTGCTAGCCTCTGGATTTGA 1 2-16-2 141923 None (scrambledCCTTCCCTGAAGGTTCCTCC 2 5-10-5 117405 TRADD GCTCATACTCGTAGGCCA 3 4-10-4325589 TRADD GCTCATACTCGTAGGCCA 3 5-8-5 325590 TRADD GCTCATACTCGTAGGCCA3 6-6-6  29837 None (scrambled TCGATCTCCTTTTATGCCCG 4 5-10-5 325593mTRADD CGCTCATACTCGTAGGCCAG 112 3-10-7 325594 TRADD CGCTCATACTCGTAGGCCAG112 7-10-3 325584 TRADD CGCTCATACTCGTAGGCCAG 112 5-10-5 113715 PTP1BGCTCCTTCCACTGATCCTGC 113 5-10-5 344177 PTP1B GCTCCTTCCACTGATCCTGC 1133-14-3 372350 GCCR TCTGTCTCTCCCATATACAG 5 2-16-2 372376 GCCRTGTTTCTGTCTCTCCCATAT 6 2-16-2 372331 GCCR CTTTTGTTTCTGTCTCTCCC 7 2-16-2372341 GCCR ATCACTTTTGTTTCTGTCTC 8 2-16-2 352983 GCCRGTTTGCAATGCTTTCTTCCA 9 2-16-2 372365 GCCR TGAGGTTTGCAATGCTTTCT 10 2-16-2372387 GCCR CTATTGAGGTTTGCAATGCT 11 2-16-2 372316 GCCRCGACCTATTGAGGTTTGCAA 12 2-16-2 372310 GCCR CTGGTCGACCTATTGAGGTT 132-16-2 372315 GCCR CTGTGGTATACAATTTCACA 14 2-16-2 372326 GCCRCTTTGGTCTGTGGTATACAA 15 2-16-2 372339 GCCR GTCAAAGGTGCTTTGGTCTG 162-16-2 372322 GCCR GGTTTAGTGTCCGGTAAAAT 17 2-16-2 372361 GCCRCTTTTTCTGTTTTCACTTGG 18 2-16-2 372308 GCCR TTCTCTTGCTTAATTACCCC 192-16-2 372304 GCCR CAGTTTCTCTTGCTTAATTA 20 2-16-2 352984 GCCRGCCCAGTTTCTCTTGCTTAA 21 2-16-2 372372 GCCR TTTATTACCAATTATATTTG 222-16-2 372327 GCCR ACATTTTATTACCAATTATA 23 2-16-2 372311 GCCRGCAGACATTTTATTACCAAT 24 2-16-2 372352 GCCR AATGGCAGACATTTTATTAC 252-16-2 372337 GCCR CAGAAATGGCAGACATTTTA 26 2-16-2 372323 GCCRTGAACAGAAATGGCAGACAT 27 2-16-2 372347 GCCR CCATGAACAGAAATGGCAGA 282-16-2 372383 GCCR CACACCATGAACAGAAATGG 29 2-16-2 372348 GCCRTACTCACACCATGAACAGAA 30 2-16-2 372363 GCCR GAGGTACTCACACCATGAAC 312-16-2 372334 GCCR TCCAGAGGTACTCACACCAT 32 2-16-2 372359 GCCRGTCCTCCAGAGGTACTCACA 33 2-16-2 372344 GCCR ATCTGTCCTCCAGAGGTACT 342-16-2 372307 GCCR GTACATCTGTCCTCCAGAGG 35 2-16-2 372370 GCCRAGTGGTACATCTGTCCTCCA 36 2-16-2 372374 GCCR TCATAGTGGTACATCTGTCC 372-16-2 372355 GCCR CATGTCATAGTGGTACATCT 38 2-16-2 372385 GCCRTATTCATGTCATAGTGGTAC 39 2-16-2 372319 GCCR GCTGTATTCATGTCATAGTG 402-16-2 372366 GCCR GGATGCTGTATTCATGTCAT 41 2-16-2 372330 GCCRAAAGGGATGCTGTATTCATG 42 2-16-2 372333 GCCR TGAGAAAGGGATGCTGTATT 432-16-2 372358 GCCR TGGTGGAATGACATTAAAAA 44 2-16-2 372381 GCCRGAATTGGTGGAATGACATTA 45 2-16-2 372377 GCCR GAGCTTACATCTGGTCTCAT 462-16-2 372309 GCCR AGGAGAGCTTACATCTGGTC 47 2-16-2 372388 GCCRATGGAGGAGAGCTTACATCT 48 2-16-2 372321 GCCR CTGGATGGAGGAGAGCTTAC 492-16-2 372312 GCCR GAGCTGGATGGAGGAGAGCT 50 2-16-2 372324 GCCRTGTCCTTCCACTGCTCTTTT 51 2-16-2 372332 GCCR GTGCTGTCCTTCCACTGCTC 522-16-2 372335 GCCR AATTGTGCTGTCCTTCCACT 53 2-16-2 372342 GCCRAGGTAATTGTGCTGTCCTTC 54 2-16-2 372345 GCCR CGGCATGCTGGGCAGTTTTT 552-16-2 372356 GCCR ATAGCGGCATGCTGGGCAGT 56 2-16-2 372305 GCCRCGATAGCGGCATGCTGGGCA 57 2-16-2 372367 GCCR ATTCCAGCCTGAAGACATTT 582-16-2 372353 GCCR GTTCATTCCAGCCTGAAGAC 59 2-16-2 372364 GCCRTTCTTTGTTTTTCGAGCTTC 60 2-16-2 372340 GCCR TTTTTTCTTTGTTTTTCGAG 612-16-2 372369 GCCR CAGGAACTATTGTTTTGTTA 62 2-16-2 372378 GCCRTGCAGGAACTATTGTTTTGT 63 2-16-2 372317 GCCR GAGCTATCATATCCTGCATA 642-16-2 372351 GCCR AACAGAGCTATCATATCCTG 65 2-16-2 372389 GCCRCTGGAACAGAGCTATCATAT 66 2-16-2 372362 GCCR TTCACTGCTGCAATCACTTG 672-16-2 372328 GCCR CCATTTCACTGCTGCAATCA 68 2-16-2 372338 GCCRTTGCCCATTTCACTGCTGCA 69 2-16-2 372349 GCCR ATAATCAGATCAGGAGCAAA 702-16-2 372373 GCCR ATTAATAATCAGATCAGGAG 71 2-16-2 372360 GCCRGCTCATTAATAATCAGATCA 72 2-16-2 372384 GCCR CTCTGCTCATTAATAATCAG 732-16-2 372380 GCCR CATTCTCTGCTCATTAATAA 74 2-16-2 372320 GCCRAGCATGTGTTTACATTGGTC 75 2-16-2 372371 GCCR AAGGTTTTCATACAGAGATA 762-16-2 372382 GCCR CAGTAAGGTTTTCATACAGA 77 2-16-2 372306 GCCRGAAGCAGTAAGGTTTTCATA 78 2-16-2 372343 GCCR GAGAGAAGCAGTAAGGTTTT 792-16-2 372313 GCCR GCTTTTCCTAGCTCTTTGAT 80 2-16-2 372325 GCCRATGGCTTTTCCTAGCTCTTT 81 2-16-2 372336 GCCR ATGGTCTTATCCAAAAATGT 822-16-2 372318 GCCR ACTCATGGTCTTATCCAAAA 83 2-16-2 372375 GCCRCAATACTCATGGTCTTATCC 84 2-16-2 372346 GCCR AATTCAATACTCATGGTCTT 852-16-2 372386 GCCR ATGATTTCAGCTAACATCTC 86 2-16-2 372354 GCCRGTGATGATTTCAGCTAACAT 87 2-16-2 372357 GCCR GAATATTTTGGTATCTGATT 882-16-2 372368 GCCR ATTTGAATATTTTGGTATCT 89 2-16-2 372379 GCCRTTCCATTTGAATATTTTGGT 90 2-16-2 372390 GCCR ATATTTCCATTTGAATATTT 912-16-2 372329 GCCR TTTTTGATATTTCCATTTGA 92 2-16-2 361132 GCCRTCTGTCTCTCCCATATACAG 5 5-10-5 361133 GCCR TGTTTCTGTCTCTCCCATAT 6 5-10-5361134 GCCR CTTTTGTTTCTGTCTCTCCC 7 5-10-5 361135 GCCRATCACTTTTGTTTCTGTCTC 8 5-10-5 180272 GCCR GTTTGCAATGCTTTCTTCCA 9 5-10-5345188 GCCR TGAGGTTTGCAATGCTTTCT 10 5-10-5 361136 GCCRCTATTGAGGTTTGCAATGCT 11 5-10-5 361137 GCCR CGACCTATTGAGGTTTGCAA 125-10-5 180274 GCCR CTGGTCGACCTATTGAGGTT 13 5-10-5 180275 GCCRCTGTGGTATACAATTTCACA 14 5-10-5 180276 GCCR CTTTGGTCTGTGGTATACAA 155-10-5 345198 GCCR GTCAAAGGTGCTTTGGTCTG 16 5-10-5 180279 GCCRGGTTTAGTGTCCGGTAAAAT 17 5-10-5 361138 GCCR CTTTTTCTGTTTTCACTTGG 185-10-5 180280 GCCR TTCTCTTGCTTAATTACCCC 19 5-10-5 345218 GCCRCAGTTTCTCTTGCTTAATTA 20 5-10-5 180281 GCCR GCCCAGTTTCTCTTGCTTAA 215-10-5 361139 GCCR TTTATTACCAATTATATTTG 22 5-10-5 361140 GCCRACATTTTATTACCAATTATA 23 5-10-5 361141 GCCR GCAGACATTTTATTACCAAT 245-10-5 361142 GCCR AATGGCAGACATTTTATTAC 25 5-10-5 361143 GCCRCAGAAATGGCAGACATTTTA 26 5-10-5 361144 GCCR TGAACAGAAATGGCAGACAT 275-10-5 180283 GCCR CCATGAACAGAAATGGCAGA 28 5-10-5 361145 GCCRCACACCATGAACAGAAATGG 29 5-10-5 361146 GCCR TACTCACACCATGAACAGAA 305-10-5 361147 GCCR GAGGTACTCACACCATGAAC 31 5-10-5 361148 GCCRTCCAGAGGTACTCACACCAT 32 5-10-5 361149 GCCR GTCCTCCAGAGGTACTCACA 335-10-5 361150 GCCR ATCTGTCCTCCAGAGGTACT 34 5-10-5 361151 GCCRGTACATCTGTCCTCCAGAGG 35 5-10-5 361152 GCCR AGTGGTACATCTGTCCTCCA 365-10-5 361153 GCCR TCATAGTGGTACATCTGTCC 37 5-10-5 361154 GCCRCATGTCATAGTGGTACATCT 38 5-10-5 361155 GCCR TATTCATGTCATAGTGGTAC 395-10-5 361156 GCCR GCTGTATTCATGTCATAGTG 40 5-10-5 361157 GCCRGGATGCTGTATTCATGTCAT 41 5-10-5 361158 GCCR AAAGGGATGCTGTATTCATG 425-10-5 180288 GCCR TGAGAAAGGGATGCTGTATT 43 5-10-5 180289 GCCRTGGTGGAATGACATTAAAAA 44 5-10-5 361159 GCCR GAATTGGTGGAATGACATTA 455-10-5 361160 GCCR GAGCTTACATCTGGTCTCAT 46 5-10-5 361161 GCCRAGGAGAGCTTACATCTGGTC 47 5-10-5 361162 GCCR ATGGAGGAGAGCTTACATCT 485-10-5 361163 GCCR CTGGATGGAGGAGAGCTTAC 49 5-10-5 361164 GCCRGAGCTGGATGGAGGAGAGCT 50 5-10-5 361165 GCCR TGTCCTTCCACTGCTCTTTT 515-10-5 361166 GCCR GTGCTGTCCTTCCACTGCTC 52 5-10-5 361167 GCCRAATTGTGCTGTCCTTCCACT 53 5-10-5 361168 GCCR AGGTAATTGTGCTGTCCTTC 545-10-5 361169 GCCR CGGCATGCTGGGCAGTTTTT 55 5-10-5 361170 GCCRATAGCGGCATGCTGGGCAGT 56 5-10-5 361171 GCCR CGATAGCGGCATGCTGGGCA 575-10-5 361172 GCCR ATTCCAGCCTGAAGACATTT 58 5-10-5 361173 GCCRGTTCATTCCAGCCTGAAGAC 59 5-10-5 361174 GCCR TTCTTTGTTTTTCGAGCTTC 605-10-5 361175 GCCR TTTTTTCTTTGTTTTTCGAG 61 5-10-5 180297 GCCRCAGGAACTATTGTTTTGTTA 62 5-10-5 361176 GCCR TGCAGGAACTATTGTTTTGT 635-10-5 361177 GCCR GAGCTATCATATCCTGCATA 64 5-10-5 361178 GCCRAACAGAGCTATCATATCCTG 65 5-10-5 361179 GCCR CTGGAACAGAGCTATCATAT 665-10-5 361180 GCCR TTCACTGCTGCAATCACTTG 67 5-10-5 361181 GCCRCCATTTCACTGCTGCAATCA 68 5-10-5 361182 GCCR TTGCCCATTTCACTGCTGCA 695-10-5 361183 GCCR ATAATCAGATCAGGAGCAAA 70 5-10-5 361184 GCCRATTAATAATCAGATCAGGAG 71 5-10-5 361185 GCCR GCTCATTAATAATCAGATCA 725-10-5 361186 GCCR CTCTGCTCATTAATAATCAG 73 5-10-5 180302 GCCRCATTCTCTGCTCATTAATAA 74 5-10-5 180304 GCCR AGCATGTGTTTACATTGGTC 755-10-5 361187 GCCR AAGGTTTTCATACAGAGATA 76 5-10-5 361188 GCCRCAGTAAGGTTTTCATACAGA 77 5-10-5 361189 GCCR GAAGCAGTAAGGTTTTCATA 785-10-5 180307 GCCR GAGAGAAGCAGTAAGGTTTT 79 5-10-5 361190 GCCRGCTTTTCCTAGCTCTTTGAT 80 5-10-5 361191 GCCR ATGGCTTTTCCTAGCTCTTT 815-10-5 361192 GCCR ATGGTCTTATCCAAAAATGT 82 5-10-5 361193 GCCRACTCATGGTCTTATCCAAAA 83 5-10-5 361194 GCCR CAATACTCATGGTCTTATCC 845-10-5 361195 GCCR AATTCAATACTCATGGTCTT 85 5-10-5 361196 GCCRATGATTTCAGCTAACATCTC 86 5-10-5 180311 GCCR GTGATGATTTCAGCTAACAT 875-10-5 361197 GCCR GAATATTTTGGTATCTGATT 88 5-10-5 361198 GCCRATTTGAATATTTTGGTATCT 89 5-10-5 361199 GCCR TTCCATTTGAATATTTTGGT 905-10-5 361200 GCCR ATATTTCCATTTGAATATTT 91 5-10-5 361202 GCCRTTTTTGATATTTCCATTTGA 92 5-10-5 310457 GCGR GCACTTTGTGGTGCCAAGGC 935-10-5 325448 GCGR GCACTTTGTGGTGCCAAGGC 93 2-16-2 325568 GCGRGCACTTTGTGGTGCCAAGGC 93 3-14-3 356171 GCGR GCACTTTGTGGTACCAAGGT 945-10-5 357368 GCGR GCACTTTGTGGTACCAAGGT 94 Uniform deoxy 357369 GCGRGCACTTTGTGGTACCAAGGT 94 1-18-1 357370 GCGR GCACTTTGTGGTACCAAGGT 941-17-2 357371 GCGR GCACTTTGTGGTACCAAGGT 94 2-16-2 357372 GCGRGCACTTTGTGGTACCAAGGT 94 3-14-3 357373 GCGR GCACTTTGTGGTACCAAGGT 944-12-4 217328 DGAT2 GCATTGCCACTCCCATTCTT 95 5-10-5 334177 DGAT2AGGACCCCGGAGTAGGCGGC 96 5-10-5 366710 DGAT2 GACCTATTGAGCCAGGTGAC 975-10-5 366714 DGAT2 GTAGCTGCTTTTCCACCTTG 98 5-10-5 370727 DGAT2AGCTGCTTTTCCACCTTGGA 99 2-16-2 370747 DGAT2 TGGAGCTCAGAGACTCAGCC 1002-16-2 370784 DGAT2 GCTGCATCCATGTCATCAGC 101 2-16-2

TABLE 2 Target sequences Target name Synonyms Species GENBANK AccessionNo or description SEQ ID NO PTEN MMAC1; TEP1; TGF beta regulated andmouse U92437.1 103 epithelial cell-enriched phosphatase; mutated inmultiple advanced cancers 1; phosphatase and tensin homologue; putativeprotein tyrosine phosphatase TRADD TNF receptor 1 associated protein;mouse consensus sequence built from mouse ESTs: 104 TNFRSF1A-associatedvia death domain; aa013629, aa914725, aa013699, aa122508, aa881900,Tumor necrosis factor receptor associated aa423244, aa930854, w13708,aa201054, ai122320, death domain aa611848, aa546092, and aa939422 GCCRnuclear receptor subfamily 3, group C, human NM_000176.1 105 member 1;GR; GRL; NR3C1; rat NM_012576.1 106 glucocorticoid receptor; nuclearreceptor mouse NM_008173.1 107 subfamily 3, group C, member 1 GCGRglucagon receptor; GR human NM_000160.1 108 rat M96674.1 109 DGAT2ACYL-CoA: DIACYLGLYCEROL human NM_032564.2 110 ACYLTRANSFERASE 2;diacylglycerol rat the complement of nucleotides 15333000 to 111acyltransferase 2; DIACYLGLYCEROL O- 15365000 of GENBANK ® accessionnumber ACYLTRANSFERASE 2; GS1999full; NW_047561.1 LOC84649 PTP1B PTP-1B;PTPN1; RKPTP; protein tyrosine human M31724.1 114 phosphatase; proteintyrosine phosphatase 1B; protein tyrosine phosphatase, non- receptortype 1

Example 2 Assaying Modulation of Expression

Modulation of target RNA expression can be assayed in a variety of waysknown in the art. GCCR mRNA levels can be quantitated by, e.g., Northernblot analysis, competitive polymerase chain reaction (PCR), or real-timePCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA by methods known in the art. Methods of RNA isolation are taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons,Inc., 1993.

Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

Levels of proteins encoded by a target RNA can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), ELISA or fluorescence-activatedcell sorting (FACS). Antibodies directed to a protein encoded by atarget RNA can be identified and obtained from a variety of sources,such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham,Mich.), or can be prepared via conventional antibody generation methods.Methods for preparation of polyclonal antisera are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997.Preparation of monoclonal antibodies is taught in, for example, Ausubel,F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

The effect of oligomeric compounds of the present invention on targetnucleic acid expression can be tested in any of a variety of cell typesprovided that the target nucleic acid is present at measurable levels.The effect of oligomeric compounds of the present invention on targetnucleic acid expression can be routinely determined using, for example,PCR or Northern blot analysis. Cell lines are derived from both normaltissues and cell types and from cells associated with various disorders(e.g. hyperproliferative disorders). Cell lines derived from multipletissues and species can be obtained from American Type CultureCollection (ATCC, Manassas, Va.), the Japanese Cancer Research ResourcesBank (Tokyo, Japan), or the Centre for Applied Microbiology and Research(Wiltshire, United Kingdom).

Primary cells, or those cells which are isolated from an animal and notsubjected to continuous culture, can be prepared according to methodsknown in the art or obtained from various commercial suppliers.Additionally, primary cells include those obtained from donor humansubjects in a clinical setting (i.e. blood donors, surgical patients).

Cell Types

The effects of oligomeric compounds on target nucleic acid expressionwere tested in the following cell types:

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Institute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached approximately90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of approximately 3000 cells/well for use inoligomeric compound transfection experiments.

HepG2 Cells:

The human hepatoblastoma cell line HepG2 was obtained from the AmericanType Culture Collection (Manassas, Va.). HepG2 cells were routinelycultured in Eagle's MEM supplemented with 10% fetal bovine serum, 1 mMnon-essential amino acids, and 1 mM sodium pyruvate (Invitrogen LifeTechnologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Multiwell culture plates are prepared for cell culture bycoating with a 1:100 dilution of type 1 rat tail collagen (BDBiosciences, Bedford, Mass.) in phosphate-buffered saline. Thecollagen-containing plates were incubated at 37° C. for approximately 1hour, after which the collagen was removed and the wells were washedtwice with phosphate-buffered saline. Cells were seeded into 96-wellplates (Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at adensity of approximately 8,000 cells/well for use in oligomeric compoundtransfection experiments.

Primary Rat Hepatocytes:

Primary rat hepatocytes are prepared from Sprague-Dawley rats purchasedfrom Charles River Labs (Wilmington, Mass.) and are routinely culturedin DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,Carlsbad, Calif.), 100 units per mL penicillin, and 100 μg/mLstreptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cells areseeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences,Bedford, Mass.) at a density of approximately 4,000-6,000 cells/welltreatment with the oligomeric compounds of the invention.

Treatment with Oligomeric Compounds

When cells reached appropriate confluency, they were treated witholigonucleotide using a transfection method as described. Other suitabletransfection reagents known in the art include, but are not limited to,LIPOFECTAMINE™, CYTOFECTIN™, OLIGOFECTAMINE™, and FUGENE™. Othersuitable transfection methods known in the art include, but are notlimited to, electroporation.

LIPOFECTIN™

When cells reach 65-75% confluency, they are treated witholigonucleotide. Oligonucleotide is mixed with UPOFECTIN™ InvitrogenLife Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reduced serum medium(Invitrogen Life Technologies, Carlsbad, Calif.) to achieve the desiredconcentration of oligonucleotide and a LIPOFECTIN™ concentration of 2.5or 3 μg/mL per 100 nM oligonucleotide. This transfection mixture isincubated at room temperature for approximately 0.5 hours. For cellsgrown in 96-well plates, wells are washed once with 100 μL OPTI-MEM™-1and then treated with 130 μL of the transfection mixture. Cells grown in24-well plates or other standard tissue culture plates are treatedsimilarly, using appropriate volumes of medium and oligonucleotide.Cells are treated and data are obtained in duplicate or triplicate.After approximately 4-7 hours of treatment at 37° C., the mediumcontaining the transfection mixture is replaced with fresh culturemedium. Cells are harvested 16-24 hours after oligonucleotide treatment.

Example 3 Real-Time Quantitative PCR Analysis of GCCR mRNA Levels

Quantitation of GCCR mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions.

Gene target quantities obtained by RT, real-time PCR were normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). Total RNA was quantified using RiboGreen™RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). 170μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10mM Tris-HCl, 1 mM EDTA, pH 7.5) was pipetted into a 96-well platecontaining 30 μL purified cellular RNA. The plate was read in aCytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm andemission at 530 nm.

GAPDH expression was quantified by RT, real-time PCR, eithersimultaneously with the quantification of the target or separately. Formeasurement simultaneous with measurement of target levels, primer-probesets specific to the target gene being measured were evaluated for theirability to be “multiplexed” with a GAPDH amplification reaction prior toquantitative PCR analysis. Multiplexing refers to the detection ofmultiple DNA species, in this case the target and endogenous GAPDHcontrol, in a single tube, which requires that the primer-probe set forGAPDH does not interfere with amplification of the target.

Probes and primers for use in real-time PCR were designed to hybridizeto target-specific sequences. Methods of primer and probe design areknown in the art. Design of primers and probes for use in real-time PCRcan be carried out using commercially available software, for examplePrimer Express®, PE Applied Biosystems, Foster City, Calif. Thetarget-specific PCR probes have FAM covalently linked to the 5′ end andTAMRA or MGB covalently linked to the 3′ end, where FAM is thefluorescent dye and TAMRA or MGB is the quencher dye.

After isolation, the RNA is subjected to sequential reversetranscriptase (RT) reaction and real-time PCR, both of which areperformed in the same well. RT and PCR reagents were obtained fromInvitrogen Life Technologies (Carlsbad, Calif.). RT, real-time PCR wascarried out in the same by adding 20 μL PCR cocktail (2.5× PCR bufferminus MgCl2, 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375nM each of forward primer and reverse primer, 125 nM of probe, 4 UnitsRNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reversetranscriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL totalRNA solution (20-200 ng). The RT reaction was carried out by incubationfor 30 minutes at 48° C. Following a 10 minute incubation at 95° C. toactivate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

Example 4 Increased Potency of ISIS 344266 (2-16-2) In Vivo Compared to5-10-5 Compound is not due to Enhanced Oligonucleotide Accumulation

Mice were dosed with ISIS 116847 (SEQ ID NO: 1) or ISIS 344266 (SEQ IDNO: 1) at 6, 3, 1.5 or 0.75 micromol/kg (approx 40, 20, 10 or 5 mg perkg), twice a week for three weeks and sacrificed 48 hours after the lastdose was given. The left panel of FIG. 1 is a graph showing percentreduction of target RNA in liver following administration of saline,ISIS 141923 (negative unrelated control oligonucleotide, incorporatedherein as SEQ ID NO: 2), ISIS 116847 at four concentrations, or ISIS344266 at four concentrations. Both ISIS 116847 and ISIS 344266 aretargeted to mouse PTEN (GENBANK® Accession No: U92437.1, hereinincorporated as SEQ ID NO: 103), and are cross-species oligonucleotideswith perfect complementary to human, rat, and rabbit PTEN. Neithersaline nor negative control ISIS 141923 (6 micromoles/kg) reduced PTENRNA levels. ISIS 116847 reduced PTEN RNA levels by approximately 21%,44%, 64% and 81% at doses of 0.75, 1.5, 3 and 6 micromol/kg,respectively. ISIS 344266, the gap-widened antisense oligonucleotide,reduced PTEN RNA levels by approximately 54%, 79%, 88% and 91% at dosesof 0.75, 1.5, 3 and 6 micromol/kg, respectively. A correspondingreduction of PTEN protein was demonstrated by Western blot as shown inthe right panel of FIG. 1.

The ID50 (dose resulting in 50% reduction of PTEN RNA) calculated fromthese results was 1.9 micromol/kg for 116847 and 0.63 micromol/kg for344266. The IC50 for ISIS 116847 was also over three-fold that of ISIS344266. These results indicate that the gap-widened antisenseoligonucleotide is three-fold more potent than the 5-10-5 compound ofequivalent sequence.

ISIS 344266 (2-16-2) supports similar persistence of action compared toISIS 116847 (5-10-5). Mice were treated as described above with ISIS344266 (1.5 or 6 micromol/kg) or ISIS 116847 (6 micromol/kg), or withsaline. PTEN RNA levels were measured in mouse liver at days 1, 7, 14and 28. As shown in FIG. 2, the two compounds show similar durability ofreduction of PTEN RNA levels, and even after 28 days the PTEN RNA levelsin antisense-treated animals (either 116847 or 344266) had not returnedto control levels.

The advantage conveyed by the gap-widened antisense oligonucleotides ofthe present invention for target reduction in vivo is surprising becauseit is not observed in vitro. An in vitro comparison of the same PTENoligonucleotides, ISIS 116847 (5-10-5) and ISIS 344266 (2-16-2) wasperformed in cultured mouse bEND cells. Cells were transfected witholigonucleotide at doses of 0.1 nM, 0.3 nM, 0.9 nM, 2.7 nM, 8.1 nM and24.3 nM in the presence of 3 microgram/ml LIPOFECTIN. Reduction oftarget expression was assayed by quantitative RT real-time PCR asdescribed herein. FIG. 3 shows that the 5-10-5 gapmer was less potentthan the 2-16-2 gapmer. The IC50 for reduction of PTEN RNA by the 5-10-5gapmer (ISIS 116847) was 3.4 nM and 6.2 nM for the 2-16-2 gapmer (ISIS344266). Thus the advantage conveyed by the gap-widened antisenseoligonucleotides for target reduction in liver is not observed incultured cells.

The enhanced potency of the gap-widened (2-16-2) PTEN antisenseoligonucleotide in liver is not due to increased concentrations in livercompared to the 5-10-5 gapmer. Oligonucleotide concentration in kidneyand liver tissue from mice treated as described above with ISIS 116847or ISIS 344266 were determined. Methods to determine oligonucleotideconcentration in tissues are known in the art (Geary et al., AnalBiochem, 1999, 274, 241-248). Oligonucleotide concentrations(micrograms/gram) in mouse liver and kidney were determined. As shown inFIG. 4, there was consistently less ISIS 344266 than ISIS 116847 inliver at every oligonucleotide dosage. The same is true for kidneyalthough overall concentrations of both compounds were lower in kidney.Thus, the enhanced potency of the gap-widened antisense oligonucleotide(2-16-2 chimera) in the liver is not due to enhanced accumulation ofcompound in the liver.

Serum transaminases (AST/ALT) were higher for mice treated with 2-16-2compound (ISIS 344266) than for those treated with ISIS 116847. However,because ISIS 344266 is more potent (active at lower doses), thetherapeutic window for the two compounds is roughly comparable.

Example 5 Effect of Gap Size on In Vitro and In Vivo Potency

A series of MOE gapmers (2-14-2 through 6-6-6) were designed to targetmouse TRADD (consensus sequence built from mouse ESTs:aa013629,aa914725,aa013699, aa122508, aa881900, aa423244, aa930854,w13708, aa201054, ai122320, aa611848, aa546092, and aa939422,incoporated herein as SEQ ID NO: 104). As shown in Table 2, a series of18 mer chimeric antisense oligonucleotides were synthesized, all havingthe same sequence (GCTCATACTCGTAGGCCA, incorporated herein as SEQ ID NO:3). Plain text indicates a deoxynucleotide, and nucleobases designatedwith bold, underlined text are 2′-O-(2-methoxyethyl)nucleotides.Internucleoside linkages are phosphorothioate throughout, and allcytosines are 5-methylcytosines. Indicated in Table 2 is the “motif' ofeach compound indicative of chemically distinct regions comprising theoligonucleotide.

TABLE 2 Antisense oligonucleotides targeting mouse TRADD ISIS NumberChemistry Motif ISIS 325589 GCTCA TACTCGTA GGCCA 5-8-5 ISIS 117405 GCTCATACTCGTAG GCCA 4-10-4 ISIS 325588 GCT CATACTCGTAGG CCA 3-12-3ISIS 325587 GC TCATACTCGTAGGC CA 2-14-2 ISIS 325590 GCTCAT ACTCGT AGGCCA6-6-6

The compounds were tested in vitro in mouse bEND cells at concentrationsof 0.1 nM, 0.5 nM, 2.5 nM, 12.5 nM and 62.5 nM for their ability toreduce target mRNA levels using real-time PCR as described herein. Asshown in FIG. 5, in vitro IC50s for these compounds were 9.2 nM for the5-8-5 gapmer (ISIS 325589), 11 nM for the 4-10-4 gapmer (ISIS 117405),19 nM for the 3-12-3 gapmer (ISIS 325588), 49 nM for the 2-142 gapmer(ISIS 325587) and 82 nM for the 6-6-6 gapmer (ISIS 325590). Thus in thisin vitro experiment, larger gaps did not appear to convey added potency.

When these compounds were tested in vivo, a different rank order potencywas observed. Mice were treated with TRADD gapmer oligos (describedabove) ranging from 2-14-2 chimeras to 6-6-6 chimeras, each at doses of1.56 micromole/kg, 3.12 micromol/kg and 6.24 micromol/kg. The negativecontrol was ISIS 29837 (SEQ ID NO: 4) and animals treated with salinealone served as the control group to which data were normalized. Asshown in FIG. 6, potency in liver increased with increasing gap size(from 6 to 14 contiguous deoxynucleotides). In a subsequent experiment(not shown) the 2-14-2 compound was approximately two-fold better thanthe 4-10-4 compound.

The effect of these gapmer compounds on mouse body weight, liver weightand spleen weights was compared and no meaningful differences were seen.Mice gained weight at roughly the same rate (indicating general goodhealth) and liver and spleen weights were comparable to saline in allthe treatment groups.

Example 6 Antisense Inhibition of Human GCCR Expression by 5-10-5Gapmers or 2-16-2 Gapmers In Vitro

A series of oligomeric compounds was designed to target differentregions of human GCCR, using published sequences (GENBANK® accession no:NM_(—)000176.1, incoporated herein as SEQ ID NO: 105). The compounds areshown in Table 3. All compounds in Table 3 are chimeric oligonucleotides(”gapmers“) 20 nucleotides in length, composed of a central “gap” regionconsisting of 10 2′-deoxynucleotides, which is flanked on both sides (5′and 3′) by five-nucleotide “wings”. The wings are composed of2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOE nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate throughout theoligonucleotide. All cytidine residues are 5-methylcytidines. Shown inTable 3 is the sequence of the oligonucleotide, and the target sitewhich is the first (5′ most) position on the target sequence to whichthe compound binds. The compounds were analyzed for their effect on genetarget mRNA levels by quantitative real-time PCR as described in otherexamples herein, using a primer-probe set designed to hybridize to humanGCCR.

Data are averages from three experiments in which HepG2 cells weretreated with 50 nM of the disclosed oligomeric compounds usingLIPOFECTIN™. A reduction in expression is expressed as percentinhibition in Table 3. If present, “N.D.” indicates “not determined”.The target regions to which these oligomeric compounds are inhibitoryare herein referred to as “validated target segments.”

TABLE 3 Inhibition of human GCCR mRNA levels by 5-10-5 gapmers ISISTarget SEQ No of SEQ ID Target % Inhib ID 5-10-5 NO Site Sequencew/5-10-5 NO 361132 105  394 TCTGTCTCTCCCATATACAG 65  5 361133 105  398TGTTTCTGTCTCTCCCATAT 56  6 361134 105  402 CTTTTGTTTCTGTCTCTCCC 60  7361135 105  406 ATCACTTTTGTTTCTGTCTC 80  8 180272 105  497GTTTGCAATGCTTTCTTCCA 74  9 345188 105  501 TGAGGTTTGCAATGCTTTCT 71 10361136 105  505 CTATTGAGGTTTGCAATGCT 10 11 361137 105  509CGACCTATTGAGGTTTGCAA 80 12 180274 105  514 CTGGTCGACCTATTGAGGTT 68 13180275 105  672 CTGTGGTATACAATTTCACA 44 14 180276 105  679CTTTGGTCTGTGGTATACAA 78 15 345198 105  689 GTCAAAGGTGCTTTGGTCTG 79 16180279 105  877 GGTTTAGTGTCCGGTAAAAT 60 17 361138 105  954CTTTTTCTGTTTTCACTTGG 70 18 180280 105 1000 TTCTCTTGCTTAATTACCCC 77 19345218 105 1004 CAGTTTCTCTTGCTTAATTA 67 20 180281 105 1007GCCCAGTTTCTCTTGCTTAA 74 21 361139 105 1058 TTTATTACCAATTATATTTG  0 22361140 105 1062 ACATTTTATTACCAATTATA 35 23 361141 105 1066GCAGACATTTTATTACCAAT 78 24 361142 105 1070 AATGGCAGACATTTTATTAC 40 25361143 105 1074 CAGAAATGGCAGACATTTTA 63 26 361144 105 1078TGAACAGAAATGGCAGACAT 61 27 180283 105 1081 CCATGAACAGAAATGGCAGA 69 28361145 105 1085 CACACCATGAACAGAAATGG 30 29 361146 105 1089TACTCACACCATGAACAGAA 60 30 361147 105 1093 GAGGTACTCACACCATGAAC 71 31361148 105 1097 TCCAGAGGTACTCACACCAT 75 32 361149 105 1101GTCCTCCAGAGGTACTCACA 69 33 361150 105 1105 ATCTGTCCTCCAGAGGTACT 53 34361151 105 1109 GTACATCTGTCCTCCAGAGG 75 35 361152 105 1113AGTGGTACATCTGTCCTCCA 62 36 361153 105 1117 TCATAGTGGTACATCTGTCC 52 37361154 105 1121 CATGTCATAGTGGTACATCT 57 38 361155 105 1125TATTCATGTCATAGTGGTAC 41 39 361156 105 1129 GCTGTATTCATGTCATAGTG 67 40361157 105 1133 GGATGCTGTATTCATGTCAT 67 41 361158 105 1137AAAGGGATGCTGTATTCATG 45 42 180288 105 1141 TGAGAAAGGGATGCTGTATT 62 43180289 105 1181 TGGTGGAATGACATTAAAAA 54 44 361159 105 1185GAATTGGTGGAATGACATTA 24 45 361160 105 1324 GAGCTTACATCTGGTCTCAT 59 46361161 105 1328 AGGAGAGCTTACATCTGGTC 65 47 361162 105 1332ATGGAGGAGAGCTTACATCT 18 48 361163 105 1336 CTGGATGGAGGAGAGCTTAC 50 49361164 105 1339 GAGCTGGATGGAGGAGAGCT 49 50 361165 105 1468TGTCCTTCCACTGCTCTTTT 61 51 361166 105 1472 GTGCTGTCCTTCCACTGCTC 65 52361167 105 1476 AATTGTGCTGTCCTTCCACT 62 53 361168 105 1480AGGTAATTGTGCTGTCCTTC 52 54 361169 105 1543 CGGCATGCTGGGCAGTTTTT 78 55361170 105 1547 ATAGCGGCATGCTGGGCAGT 58 56 361171 105 1549CGATAGCGGCATGCTGGGCA 65 57 361172 105 1570 ATTCCAGCCTGAAGACATTT 24 58361173 105 1574 GTTCATTCCAGCCTGAAGAC 52 59 361174 105 1597TTCTTTGTTTTTCGAGCTTC 62 60 361175 105 1601 TTTTTTCTTTGTTTTTCGAG 48 61180297 105 1680 CAGGAACTATTGTTTTGTTA 33 62 361176 105 1682TGCAGGAACTATTGTTTTGT 46 63 361177 105 1765 GAGCTATCATATCCTGCATA 71 64361178 105 1769 AACAGAGCTATCATATCCTG 51 65 361179 105 1773CTGGAACAGAGCTATCATAT 67 66 361180 105 1840 TTCACTGCTGCAATCACTTG 52 67361181 105 1844 CCATTTCACTGCTGCAATCA 55 68 361182 105 1848TTGCCCATTTCACTGCTGCA 70 69 361183 105 1999 ATAATCAGATCAGGAGCAAA 36 70361184 105 2003 ATTAATAATCAGATCAGGAG 10 71 361185 105 2007GCTCATTAATAATCAGATCA 43 72 361186 105 2011 CTCTGCTCATTAATAATCAG  0 73180302 105 2015 CATTCTCTGCTCATTAATAA 23 74 180304 105 2053AGCATGTGTTTACATTGGTC 73 75 361187 105 2119 AAGGTTTTCATACAGAGATA 38 76361188 105 2123 CAGTAAGGTTTTCATACAGA 22 77 361189 105 2127GAAGCAGTAAGGTTTTCATA 46 78 180307 105 2131 GAGAGAAGCAGTAAGGTTTT 32 79361190 105 2212 GCTTTTCCTAGCTCTTTGAT 74 80 361191 105 2215ATGGCTTTTCCTAGCTCTTT 68 81 361192 105 2347 ATGGTCTTATCCAAAAATGT 63 82361193 105 2351 ACTCATGGTCTTATCCAAAA 66 83 361194 105 2355CAATACTCATGGTCTTATCC 54 84 361195 105 2359 AATTCAATACTCATGGTCTT 69 85361196 105 2383 ATGATTTCAGCTAACATCTC  1 86 180311 105 2386GTGATGATTTCAGCTAACAT 59 87 361197 105 2407 GAATATTTTGGTATCTGATT 59 88361198 105 2411 ATTTGAATATTTTGGTATCT 20 89 361199 105 2415TTCCATTTGAATATTTTGGT 65 90 361200 105 2419 ATATTTCCATTTGAATATTT 51 91361202 105 2425 TTTTTGATATTTCCATTTGA 20 92

Gap-widened oligonucleotides having the same sequences as the compoundsdescribed in Table 4 were also tested. All compounds in Table 4 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of 16 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′) by two-nucleotide “wings”. The wingsare composed of 2′-O-(2-methoxyethyl)nucleotides, also known as 2′-MOEnucleotides. The internucleoside (backbone) linkages arephosphorothioate throughout the oligonucleotide. All cytidine residuesare 5-methylcytidines. Shown in Table 4 is the sequence of theoligonucleotide, and the target site which is the first (5′ most)position on the target sequence to which the compound binds. The 2-16-2motif compounds were analyzed for their effect on gene target mRNAlevels by quantitative real-time PCR as described herein.

Data are averages from three experiments in which HepG2 cells weretreated with 50 nM of the disclosed oligomeric compounds usingLIPOFECTIN™. A reduction in expression is expressed as percentinhibition in Table 4. If present, “N.D.” indicates “not determined”.The target regions to which these oligomeric compounds are inhibitoryare herein referred to as “validated target segments.”

TABLE 4 Inhibition of human GCCR mRNA levels by 2-16-2 gapmers ISISTarget % SEQ No of SEQ ID Target Inhib ID 2-16-2 NO Site Sequencew/2-16-2 NO 372350 105  394 TCTGTCTCTCCCATATACAG 69  5 372376 105  398TGTTTCTGTCTCTCCCATAT 72  6 372331 105  402 CTTTTGTTTCTGTCTCTCCC 67  7372341 105  406 ATCACTTTTGTTTCTGTCTC 63  8 352983 105  497GTTTGCAATGCTTTCTTCCA 64  9 372365 105  501 TGAGGTTTGCAATGCTTTCT 69 10372387 105  505 CTATTGAGGTTTGCAATGCT 70 11 372316 105  509CGACCTATTGAGGTTTGCAA 73 12 372310 105  514 CTGGTCGACCTATTGAGGTT 70 13372315 105  672 CTGTGGTATACAATTTCACA 35 14 372326 105  679CTTTGGTCTGTGGTATACAA 54 15 372339 105  689 GTCAAAGGTGCTTTGGTCTG 81 16372322 105  877 GGTTTAGTGTCCGGTAAAAT 78 17 372361 105  954CTTTTTCTGTTTTCACTTGG 70 18 372308 105 1000 TTCTCTTGCTTAATTACCCC 84 19372304 105 1004 CAGTTTCTCTTGCTTAATTA 66 20 352984 105 1007GCCCAGTTTCTCTTGCTTAA 80 21 372372 105 1058 TTTATTACCAATTATATTTG 0 22372327 105 1062 ACATTTTATTACCAATTATA 11 23 372311 105 1066GCAGACATTTTATTACCAAT 65 24 372352 105 1070 AATGGCAGACATTTTATTAC 54 25372337 105 1074 CAGAAATGGCAGACATTTTA 36 26 372323 105 1078TGAACAGAAATGGCAGACAT 73 27 372347 105 1081 CCATGAACAGAAATGGCAGA 86 28372383 105 1085 CACACCATGAACAGAAATGG 73 29 372348 105 1089TACTCACACCATGAACAGAA 82 30 372363 105 1093 GAGGTACTCACACCATGAAC 47 31372334 105 1097 TCCAGAGGTACTCACACCAT 82 32 372359 105 1101GTCCTCCAGAGGTACTCACA 69 33 372344 105 1105 ATCTGTCCTCCAGAGGTACT 72 34372307 105 1109 GTACATCTGTCCTCCAGAGG 74 35 372370 105 1113AGTGGTACATCTGTCCTCCA 69 36 372374 105 1117 TCATAGTGGTACATCTGTCC 0 37372355 105 1121 CATGTCATAGTGGTACATCT 65 38 372385 105 1125TATTCATGTCATAGTGGTAC 18 39 372319 105 1129 GCTGTATTCATGTCATAGTG 23 40372366 105 1133 GGATGCTGTATTCATGTCAT 37 41 372330 105 1137AAAGGGATGCTGTATTCATG 80 42 372333 105 1141 TGAGAAAGGGATGCTGTATT 68 43372358 105 1181 TGGTGGAATGACATTAAAAA 67 44 372381 105 1185GAATTGGTGGAATGACATTA 30 45 372377 105 1324 GAGCTTACATCTGGTCTCAT 45 46372309 105 1328 AGGAGAGCTTACATCTGGTC 63 47 372388 105 1332ATGGAGGAGAGCTTACATCT 55 48 372321 105 1336 CTGGATGGAGGAGAGCTTAC 51 49372312 105 1339 GAGCTGGATGGAGGAGAGCT 60 50 372324 105 1468TGTCCTTCCACTGCTCTTTT 73 51 372332 105 1472 GTGCTGTCCTTCCACTGCTC 81 52372335 105 1476 AATTGTGCTGTCCTTCCACT 42 53 372342 105 1480AGGTAATTGTGCTGTCCTTC 100 54 372345 105 1543 CGGCATGCTGGGCAGTTTTT 82 55372356 105 1547 ATAGCGGCATGCTGGGCAGT 73 56 372305 105 1549CGATAGCGGCATGCTGGGCA 80 57 372367 105 1570 ATTCCAGCCTGAAGACATTT 78 58372353 105 1574 GTTCATTCCAGCCTGAAGAC 70 59 372364 105 1597TTCTTTGTTTTTCGAGCTTC 47 60 372340 105 1601 TTTTTTCTTTGTTTTTCGAG 100 61372369 105 1680 CAGGAACTATTGITTTGTTA 56 62 372378 105 1682TGCAGGAACTATTGTTTTGT 41 63 372317 105 1765 GAGCTATCATATCCTGCATA 84 64372351 105 1769 AACAGAGCTATCATATCCTG 69 65 372389 105 1773CTGGAACAGAGCTATCATAT 76 66 372362 105 1840 TTCACTGCTGCAATCACTTG 64 67372328 105 1844 CCATTTCACTGCTGCAATCA 81 68 372338 105 1848TTGCCCATTTCACTGCTGCA 82 69 372349 105 1999 ATAATCAGATCAGGAGCAAA 10 70372373 105 2003 ATTAATAATCAGATCAGGAG 30 71 372360 105 2007GCTCATTAATAATCAGATCA 27 72 372384 105 2011 CTCTGCTCATTAATAATCAG 100 73372380 105 2015 CATTCTCTGCTCATTAATAA 2 74 372320 105 2053AGCATGTGTTTACATTGGTC 75 75 372371 105 2119 AAGGTTTTCATACAGAGATA 37 76372382 105 2123 CAGTAAGGTTTTCATACAGA 44 77 372306 105 2127GAAGCAGTAAGGTTTTCATA 48 78 372343 105 2131 GAGAGAAGCAGTAAGGTTTT 46 79372313 105 2212 GCTTTTCCTAGCTCTTTGAT 66 80 372325 105 2215ATGGCTTTTCCTAGCTCTTT 69 81 372336 105 2347 ATGGTCTTATCCAAAAATGT 65 82372318 105 2351 ACTCATGGTCTTATCCAAAA 70 83 372375 105 2355CAATACTCATGGTCTTATCC 85 84 372346 105 2359 AATTCAATACTCATGGTCTT 47 85372386 105 2383 ATGATTTCAGCTAACATCTC 74 86 372354 105 2386GTGATGATTTCAGCTAACAT 66 87 372357 105 2407 GAATATTTTGGTATCTGATT 13 88372368 105 2411 ATTTGAATATTTTGGTATCT 0 89 372379 105 2415TTCCATTTGAATATTTTGGT 44 90 372390 105 2419 ATATTTCCATTTGAATATTT 0 91372329 105 2425 TTTTTGATATTTCCATTTGA 0 92

The 2-16-2 oligonucleotides shown in Table 4 and the 5-10-5oligonucleotides shown in Table 3 which reduced GCCR expression by atleast 30% are preferred. The target segments to which these preferredsequences are complementary are herein referred to as “preferred targetsegments” and are therefore preferred for targeting by compounds of thepresent invention.

Example 7 Cross-Species Oligonucleotides Targeting GCCR

Some oligonucleotides described in the previous example arecomplementary across species and are therefore expected to reduceexpression of glucocorticoid receptor across species. Shown in Table 5is the sequence of such cross-species oligonucleotides, and the ISISnumbers of the 5-10-5 motif version and the 2-16-2 motif version of theoligonucleotide. Also indicated for each sequence is the target sitewhich is the first (5′ most) position on the human target sequence(NM_(—)000176.1, incorporated herein as SEQ ID NO: 105) to which thecompound binds. The complementarity for human, cynomolgus monkey, rat,and mouse GCCR mRNA is indicated (“yes” means perfect complementarityand “1 mm” means one mismatch from perfect complementarity).

TABLE 5 Cross-species oligonucleotides targeted to GCCR Pos'n ISIS # ofISIS # of SEQ on 5-10-5 2-16-2 ID SEQ ID Perfect complement to: gapmergapmer NO Sequence NO: 1 Human Monkey Rat Mouse 361137 372316 12cgacctattgaggtttgcaa  509 yes yes yes yes 180276 372326 15ctttggtctgtggtatacaa  679 yes 1 mm 1 mm yes 345198 372339 16gtcaaaggtgctttggtctg  689 yes yes yes yes 180304 372320 75agcatgtgtttacattggtc 2053 yes yes yes yes 180275 372315 14ctgtggtatacaatttcaca  672 yes 1 mm 1 mm yes 361141 372311 24gcagacattttattaccaat 1066 yes yes yes 1 mm 180281 352984 21gcccagtttctcttgcttaa 1007 yes yes yes yes 361151 372307 35gtacatctgtcctccagagg 1109 yes yes yes yes 180274 372310 13ctggtcgacctattgaggtt  514 yes yes yes yes 361156 372319 40gctgtattcatgtcatagtg 1129 yes yes yes yes

Example 8 Antisense Inhibition of Human and Rat GCCR mRNALevels—Dose-Response Studies with 5-10-5 Gapmers

In a further embodiment of the present invention, elevenoligonucleotides were selected for additional dose-response studies.Primary rat hepatocytes were treated with 5, 10, 25, 50, 100 or 200 nMof ISIS 180274, ISIS 180275, ISIS 180276, ISIS 180281, ISIS 180304, ISIS361137, ISIS 361141, ISIS 361151, ISIS 361156, ISIS 345198, ISIS 361137or the negative control oligonucleotide ISIS 141923(CCTTCCCTGAAGGTTCCTCC, incorporated herein as SEQ ID NO: 2), and mRNAlevels were measured as described in other examples herein. ISIS 141923is a 5-10-5 gapmer comprising a ten deoxynucleotide gap flanked by2′-MOE wings and a phosphorothioate backbone. All cytosines are5-methylcytosines. Untreated cells served as the control to which thedata were normalized.

Results of these studies are shown in Table 6. Target mRNA levels weremeasured by real-time PCR as described herein. Data are averages fromthree experiments and are expressed as percent inhibition relative tountreated control.

TABLE 6 Dose-dependent inhibition of GCCR expression in rat primaryhepatocytes % Inhibition Dose of Oligonucleotide (nM) ISIS # SEQ ID NO 510 25 50 100 200 180274 13 16 33 29 65 84 89 180275 14 0 13 56 84 84 90180276 15 23 43 43 68 89 93 180281 21 0 20 33 75 86 87 180304 75 42 5147 75 86 91 361137 12 40 30 48 81 83 89 361141 24 36 61 48 77 87 92361151 35 10 28 42 77 90 94 361156 40 22 47 46 66 84 92 345198 16 0 3553 81 77 85 361158 42 34 50 47 79 91 93 141923 2 0 10 18 43 0 12

In a further embodiment of the present invention, the sameoligonucleotides were tested in the human HepG2 cell line for theirability to reduce GCCR mRNA expression at the indicated doses. Untreatedcells served as the control to which the data were normalized.

Results of these studies are shown in Table 7. Target mRNA levels weremeasured by real-time PCR as described herein. Data are averages fromthree experiments and are expressed as percent inhibition relative tountreated control.

TABLE 7 Dose-dependent inhibition of GCCR expression in HepG2 cells %Inhibition Dose of Oligonucleotide (nM) ISIS # SEQ ID NO 1 10 25 50 100200 180274 13 0 31 54 66 77 83 180275 14 13 54 75 86 93 94 180276 15 2677 87 92 94 98 180281 21 3 46 68 80 90 84 180304 75 0 64 90 90 92 91361137 12 18 71 84 91 92 86 361141 24 1 49 81 85 73 78 361151 35 22 4271 82 89 91 361156 40 7 75 75 79 80 82 345198 16 17 71 79 86 80 82361158 42 11 35 78 80 82 77 141923 2 15 12 20 12 14 3

As shown in Table 6 and Table 7, antisense oligonucleotides targetingGCCR are effective at reducing both human and rat target mRNA levels ina dose-dependent manner in vitro.

Example 9 Antisense Inhibition of Rat GCCR mRNA Levels—In VivoDose-Response Studies with 5-10-5 Gapmers

Five of the 5-10-5 gapmer motif oligonucleotides (ISIS 180281, ISIS361137, ISIS 345198, ISIS 180304, and ISIS 361141) were evaluated atvarious doses in rats for their ability to reduce GCCR mRNA levels inliver. Eight week-old Sprague Dawley rats were divided into treatmentgroups which received doses of 50, 25 or 12.5 mg/kg of one the indicatedoligonucleotides via injection. Each treatment group was comprised offour animals, and was dosed twice weekly for 3 weeks. Animals injectedwith saline alone served as a control group. The animals were evaluatedweekly for standard blood parameters (ALT/AST, cholesterol,triglycerides, and glucose). Animals were sacrificed at the end of thestudy and liver tissue was collected and analyzed for target reductionusing real-time PCR analysis methods described herein. Results are shownin Tables 8a and 8b (separate experiments) as the percentage reductionin GCCR mRNA measured after treatment with the indicated doses of theindicated oligonucleotides.

TABLE 8a In vivo rat screen- GCCR antisense oligonucleotides % Reductionin GCCR mRNA in rat liver (compared to saline-treated controls) Compound50 mg/kg 25 mg/kg 12.5 mg/kg ISIS 180281 68 65 48 ISIS 180304 52 34 0ISIS 345198 63 58 52

TABLE 8b In vivo rat screen- GCCR antisense oligonucleotides % Reductionin GCCR mRNA in rat liver (compared to saline-treated controls) Compound50 mg/kg 25 mg/kg 12.5 mg/kg ISIS 180281 62 62 59 ISIS 361137 59 47 32ISIS 361141 61 49 22

The data in Tables 8a and 8b show that antisense oligonucleotidestargeted to GCCR are effective at reducing expression in vivo in adose-dependent manner. ISIS 345198 (GTCAAAGGTGCTTTGGTCTG; SEQ ID NO: 16)was chosen for further evaluation in structure-activity experimentsfocusing on gap optimization. This compound is perfectly complementaryto mouse, rat, human, monkey, rabbit and guinea pig glucocorticoidreceptor RNA.

Example 10 Antisense Inhibition of GCCR mRNA Levels In Vivo—GapOptimization Study

A series of oligomeric compounds were designed to target GCCR withvarying sizes of the deoxynucleotide gap and 2′-MOE wings. Each of theoligonucleotides tested has the same nucleobase sequence(GTCAAAGGTGCTTTGGTCTG, incorporated herein as SEQ ID NO: 16) andtherefore targets the same segment of SEQ ID NO: 105 (nucleobases 689 to709). As shown in Example 7, this oligonucleotide is also perfectlycomplementary to rat GCCR.

The compounds are shown in Table 9. Plain text indicates adeoxynucleotide, and nucleobases designated with bold, underlined textare 2′-O-(2-methoxyethyl)nucleotides. Internucleoside linkages arephosphorothioate throughout, and all cytosines are 5-methylcytosines.

Indicated in Table 9 is the “motif' of each compound indicative ofchemically distinct regions comprising the oligonucleotide.

TABLE 9 Antisense compounds targeting rat GCCR ISIS Number ChemistryMotif 345198 GTCAA AGGTGCTTTG GTCTG 5-10-5 gapmer 372339 GTCAAAGGTGCTTTGGTC TG 2-16-2 gapmer 377130 GTC AAAGGTGCTTTGGT CTG3-14-3 gapmer 377131 GTCA AAGGTGCTTTGG TCTG 4-12-4 gapmer

Nine-week old Sprague-Dawley male rats were treated twice weekly forthree weeks with doses of 50, 25, 12.5, and 6.25 mg/kg of theoligonucleotides presented in Table 9. Animals injected with salinealone served as controls. Each treatment group was comprised of fouranimals.

At the end of the study, animals were sacrificed, and tissues werecollected for determination of target reduction and oligonucleotideconcentration.

White adipose tissue was analyzed for target reduction using real-timePCR analysis methods described herein. Results are shown in Tables 10a,10b, and 10c (separate experiments) as the percentage reduction in GCCRmRNA measured after treatment with the indicated doses of the indicatedoligonucleotides. Tissues from animals treated with each gap-widenedoligonucleotide were assayed for target reduction alongside tissues fromanimals treated with the 5-10-5 motif oligonucleotide for comparison.

TABLE 10a In vivo reduction of GCCR levels in white adipose tissue with2-16-2 oligonucleotides % Inhibition Treatment Dose of oligonucleotide(mg/kg) group 50 25 12.5 6.25 ISIS 345198 56 26 17 7 ISIS 372339 34 0 88

TABLE 10b In vivo reduction of GCCR levels in white adipose tissue with3-14-3 oligonucleotides % Inhibition Treatment Dose of oligonucleotide(mg/kg) group 50 25 12.5 6.25 ISIS 345198 59 49 27 22 ISIS 377130 54 3721 18

TABLE 10c In vivo reduction of GCCR levels in white adipose tissue with4-12-4 oligonucleotides % Inhibition Treatment Dose of oligonucleotide(mg/kg) group 50 25 12.5 6.25 ISIS 345198 56 23 21 7 ISIS 377131 55 2315 0

Liver tissue was also analyzed for target reduction using real-time PCRanalysis methods described herein. Results are shown in Tables 11a, 11b,and 11c (separate experiments) as the percentage reduction in GCCR mRNAmeasured after treatment with the indicated doses of the indicatedoligonucleotides. Tissues from animals treated with each gap-widenedoligonucleotide were assayed for target reduction alongside tissues fromanimals treated with the 5-10-5 motif oligonucleotide for comparison.

TABLE 11a In vivo reduction of GCCR levels in liver with 2-16-2oligonucleotides % Inhibition Treatment Dose of oligonucleotide (mg/kg)group 50 25 12.5 6.25 ISIS 345198 78 77 65 51 ISIS 372339 83 77 56 44

TABLE 11b In vivo reduction of GCCR levels in liver with 3-14-3oligonucleotides % Inhibition Treatment Dose of oligonucleotide (mg/kg)group 50 25 12.5 6.25 ISIS 345198 78 80 67 54 ISIS 377130 87 78 68 43

TABLE 11c In vivo reduction of GCCR levels in liver with 4-12-4oligonucleotides % Inhibition Treatment Dose of oligonucleotide (mg/kg)group 50 25 12.5 6.25 ISIS 345198 76 75 58 49 ISIS 377131 82 64 60 61

As shown in Tables 11a, 11b, and 11c, all of the gap-widenedoligonucleotides tested were effective at reducing GCCR levels in adose-dependent manner in vivo. In addition, the gap-widenedoligonucleotides show a trend toward greater potency than the 5-10-5gapmer in the liver.

In addition, to determine effects of altering the gap size onpharmacokinetics, oligonucleotide concentration in kidney and liver weredetermined. Methods to determine oligonucleotide concentration intissues are known in the art (Geary et al., Anal Biochem, 1999, 274,241-248). Total oligonucleotide is the sum of all oligonucleotidesmetabolites detected in the tissue. Shown in Table 12 are the totalconcentration and the concentration of full length oligonucleotide (inμg/g) in the liver of animals treated with the indicated oligonucleotideat the indicated concentration.

TABLE 12 GCCR oligonucleotide concentration in rat liver Liver LiverTotal Full- Treatment Motif Dose oligo length ISIS 345198 5-10-5 25mg/kg 507 408 12.5 mg/kg 318 224 ISIS 372339 2-16-2 25 mg/kg 450 30612.5 mg/kg 311 183 ISIS 377130 3-14-3 25 mg/kg 575 315 12.5 mg/kg 350212 ISIS 377131 4-12-4 25 mg/kg 584 424 12.5 mg/kg 354 265

As shown in Table 12, the levels of full-length oligonucleotide in theliver are comparable or reduced for ISIS 372339 and ISIS 377130 ascompared to ISIS 345198. Coupled with the target reduction as shown inTable 11, these data show that the enhanced potency of the gap-widenedcompounds is not due to enhanced accumulation of the compound in theliver. Thus, preferred oligonucleotides of the present invention includegap-widened oligonucleotides that show enhanced or comparable potencywith regard to target reduction to the corresponding 5-10-5 gapmerwithout enhanced accumulation of the compound in a target tissue. Insome embodiments, the target tissue is adipose and in some embodiments,the target tissue is liver.

Example 11 Design of “Gap-Widened” Antisense Oligonucleotides TargetingHuman GCGR

A series of oligomeric compounds were designed to target human GCGR(Genbank accession number: NM_(—)000160.1, incorporated herein as SEQ IDNO: 108), with varying sizes of the deoxynucleotide gap and 2′-MOEwings. Each of the oligonucleotides is 20 nucleobases in length and hasthe same nucleobase sequence (GCACTTTGTGGTGCCAAGGC, incorporated hereinas SEQ ID NO: 93), and therefore targets the same segment of SEQ ID NO:108 (nucleobases 532 to 551). The compounds are shown in Table 13. Plaintext indicates a deoxynucleotide, and nucleotides designated with bold,underlined text are 2′-O-(2-methoxyethyl)nucleotides. Internucleosidelinkages are phosphorothioate throughout, and all cytosines are5-methylcytosines. Indicated in Table 13 is the “motif' of eachcompound, indicative of chemically distinct regions comprising theoligonucleotide.

TABLE 13 Antisense compounds targeting human GCGR ISIS Number ChemistryMotif 310457 GCACT TTGTGGTGCC AAGGC 5-10-5 gapmer 325448 GCACTTTGTGGTGCCAAG GC 2-16-2 gapmer 325568 GCA CTTTGTGGTGCCAA GGC3-14-3 gapmer

The 5-10-5 gapmer, ISIS 310457, was tested for its ability to reducetarget mRNA levels in vitro. HepG2 cells were treated with ISIS 310457using methods as described herein. ISIS 310457 was analyzed for itseffect on human glucagon receptor mRNA levels by quantitative real-timePCR and was found to reduce expression of GCGR by about 96%.

Example 12 Design of “Gap-Widened” Antisense Oligonucleotides TargetingRat GCGR

A series of oligomeric compounds were designed to target rat GCGR(Genbank accession number: M96674.1, incorporated herein as SEQ ID NO:109) with varying sizes of the deoxynucleotide gap and 2′-MOE wings.Each of the oligonucleotides tested has the same nucleobase sequence(GCACTTTGTGGTACCAAGGT, incorporated herein as SEQ ID NO: 94) andtherefore targets the same segment of SEQ ID NO: 109 (nucleobases 402 to421). The segment targeted by the rat oligonucleotides corresponds tothe segment of human GCGR targeted by ISIS 310457 (SEQ ID NO: 93). Thecompounds are shown in Table 14. Plain text indicates a deoxynucleotide,and nucleotides designated with bold, underlined text are2′-O-(2-methoxyethyl)nucleotides. Internucleoside linkages arephosphorothioate throughout, and all cytosines are 5-methylcytosines.Indicated in Table 14 is the “motif' of each compound indicative ofchemically distinct regions comprising the oligonucleotide.

TABLE 14 Antisense compounds targeting rat GCGR ISIS Number ChemistryMotif 356171 GCACT TTGTGGTACC AAGGT 5-10-5 gapmer 357368GCACTTTGTGGTACCAAGGT Uniform deoxy 357369 G CACTTTGTGGTACCAAGG T1-18-1 gapmer 357370 G CACTTTGTGGTACCAAG GT 1-17-2 gapmer 357371 GCACTTTGTGGTACCAAG GT 2-16-2 gapmer 357372 GCA CTTTGTGGTACCAA GGT3-14-3 gapmer 357373 GCAC TTTGTGGTACCA AGGT 4-12-4 gapmer

Example 13 Effects of Antisense Oligonucleotides Targeting GCGR—In VivoRat Study

In accordance with the present invention, the oligonucleotides designedto target rat GCGR were tested in vivo. Male Sprague Dawley rats, eightweeks of age, were injected with 50, 25, 12.5, or 6.25 mg/kg of ISIS356171, ISIS 357368, ISIS 357369, ISIS 357370, ISIS 357371, ISIS 357372,or ISIS 357373 twice weekly for 3 weeks for a total of 6 doses.Saline-injected animals served as a control. Each of theoligonucleotides tested has the same nucleobase sequence(GCACTTTGTGGTACCAAGGT, incorporated herein as SEQ ID NO: 94), and thechemistry and motif of each compound is described above.

After the treatment period, rats were sacrificed and target nucleic acidlevels were evaluated in liver. RNA isolation and target mRNA expressionlevel quantitation are performed as described by other examples hereinusing RIBOGREEN™. RNA from each treatment group was assayed alongsideRNA from the group treated with ISIS 356171. Results are presented inTable 15a, 15b, 15c, 15d, 15e, and 15f as a percentage of saline-treatedcontrol levels.

TABLE 15a Reduction of target levels in liver of rats treated with2-16-2 antisense oligonucleotides targeted to GCGR % Control Dose ofoligonucleotide (mg/kg) Treatment Motif 50 25 12.5 6.25 ISIS 3561715-10-5 7 20 26 36 ISIS 357371 2-16-2 11 22 35 39

TABLE 15b Reduction of target levels in liver of rats treated with3-14-3 antisense oligonucleotides targeted to GCGR % Control Dose ofoligonucleotide (mg/kg) Treatment Motif 50 25 12.5 6.25 ISIS 3561715-10-5 10 24 28 50 ISIS 357372 3-14-3 12 23 37 56

TABLE 15c Reduction of target levels in liver of rats treated with4-12-4 antisense oligonucleotides targeted to GCGR % Control Dose ofoligonucleotide (mg/kg) Treatment Motif 50 25 12.5 6.25 ISIS 3561715-10-5 10 25 36 47 ISIS 357373 4-12-4 13 22 48 47

TABLE 15d Reduction of target levels in liver of rats treated with1-17-2 antisense oligonucleotides targeted to GCGR % Control Dose ofoligonucleotide (mg/kg) Treatment Motif 50 25 12.5 6.25 ISIS 3561715-10-5 8 24 32 43 ISIS 357370 1-17-2 20 41 62 68

TABLE 15e Reduction of target levels in liver of rats treated with1-18-1 antisense oligonucleotides targeted to GCGR % Control Dose ofoligonucleotide (mg/kg) Treatment Motif 50 25 12.5 6.25 ISIS 3561715-10-5 9 27 34 46 ISIS 357369 1-18-1 33 35 58 70

TABLE 15f Reduction of target levels in liver of rats treated withuniform deoxy oligonucleotides targeted to GCGR % Control Dose ofoligonucleotide (mg/kg) Treatment Motif 50 25 12.5 6.25 ISIS 3561715-10-5 8 23 30 45 ISIS 357368 Uniform deoxy 31 43 77 73

As shown in Tables 15a, 15b, 15c, 15d, and 15e the gap-widened antisenseoligonucleotides were effective at reducing GCGR levels in vivo in adose-dependent manner.

In addition, oligonucleotide concentration in kidney and liver weredetermined. Methods to determine oligonucleotide concentration intissues are known in the art (Geary et al., Anal. Biochem., 1999, 274,241-248). Shown in Table 16 are the total oligonucleotide concentrationand the concentration of full length oligonucleotide (in μg/g) in thekidney or liver of animals treated with 25 mg/kg of the indicatedoligonucleotide. Total oligonucleotide is the sum of alloligonucleotides metabolites detected in the tissue.

TABLE 16 Concentration of oligonucleotide in liver and kidney KidneyKidney Liver Liver Total Full- Total Full- Treatment Motif oligo lengtholigo length ISIS 356171 5-10-5 gapmer 1814 1510 621 571 ISIS 356368Uniform deoxy 801 183 282 62 ISIS 356369 1-18-1 1237 475 309 171 ISIS356370 1-17-2 1127 590 370 271 ISIS 356371 2-16-2 871 515 345 253 ISIS356372 3-14-3 1149 774 497 417 ISIS 356373 4-12-4 902 687 377 326

As shown in Table 16, the concentrations of the gap-widenedoligonucleotides in kidney were generally reduced with respect to thosefound for ISIS 356171 in these tissues. Taken with the target reductiondata shown in Table 15 wherein potency was maintained with ISIS 356371,ISIS 356372, and ISIS 356373 with respect to ISIS 356171, these datasuggest that gap-widened oligos, particularly ISIS 356371, ISIS 356372,and ISIS 356373 are, in essence, more effective than ISIS 356171 atreducing target levels in the liver.

Example 14 Effects of Antisense Oligonucleotides Targeting GCGR—In VivoStudy in Cynomolgus Monkeys

To evaluate alterations in tissue distribution, potency, or therapeuticindex caused by modification of the antisense oligonucleotide motif in aprimate, cynomolgus monkeys were injected with ISIS 310457 (5-10-5motif) or ISIS 325568 (2-16-2 motif) at doses of 3, 10, or 20 mg/kg perweek. These antisense compounds show 100% complementarity to the monkeyGCGR target sequence. Animals injected with saline alone served ascontrols. The duration of the study was 7 weeks, and the animals weredosed three times during the first week, followed by once-weekly dosingfor 6 weeks. Each treatment group was comprised of 5 animals. One grouptreated with 20 mg/kg of ISIS 310457 and one group treated with 20 mg/kgof ISIS 325568 recovered for three weeks after cessation of dosing priorto sacrifice (”20 mg/kg recovery“). Other treatment groups weresacrificed at the end of the study. Liver tissues were collected toassess target reduction.

RNA isolation and target mRNA expression level quantitation wereperformed as described by other examples herein using RIBOGREEN™.Results are presented in Table 17 as a percentage of saline-treatedcontrol levels.

TABLE 17 Reduction of target levels in liver of monkeys treated withantisense oligonucleotides targeted to GCGR % Control Dose ofoligonucleotide 20 mg/kg, Treatment Motif recovery 20 mg/kg 10 mg/kg 3mg/kg ISIS 310457 5-10-5 27 34 43 71 ISIS 325568 2-16-2 43 45 54 49

As shown in Table 17, treatment with ISIS 310457 and 325568 causeddecreases in GCGR levels at all of the doses tested, and reduction intarget levels was still observed in the 20 mg/kg recovery groups. ISIS325568 caused greater reduction than ISIS 310457 at the 3 mg/kg dose.

In addition, oligonucleotide concentration in kidney and liver weredetermined. Methods to determine oligonucleotide concentration intissues are known in the art (Geary et al., Anal Biochem, 1999, 274,241-248). Shown in Table 18 are the total concentration and theconcentration of full length oligonucleotide (in μg/g) in the kidney orliver of animals treated with the indicated oligonucleotide.

TABLE 18 Concentration of oligonucleotide in liver and kidney KidneyKidney Liver Liver Total Full- Total Full- Treatment Motif Dose oligolength oligo length ISIS 310457 5-10-5  3 mg/kg 471 423 449 330 10 mg/kg1011 911 710 606 20 mg/kg 1582 1422 981 867 20 mg/kg 449 347 648 498recovery ISIS 325568 2-16-2  3 mg/kg 356 298 309 228 10 mg/kg 830 685477 339 20 mg/kg 1390 1101 739 544 20 mg/kg 264 161 344 205 recovery

As shown in Table 18, the kidney concentration of the 5-10-5 motifoligonucleotide ISIS 310457 is higher than that measured for the 2-16-2motif oligonucleotide ISIS 325568 at all concentrations tested. Takenwith the target reduction data in Table 9 for the 2-16-2 motifoligonucleotide, these data suggest that the gap-widened oligonucleotideis more potent than the corresponding 5-10-5 motif oligonucleotide,providing a more robust lowering of target mRNA levels in the liverwithout enhanced accumulation of oligonucleotide.

Example 15 Effects of Gap-Widened Oligonucleotides on Reduction of DGAT2mRNA Levels—In Vitro Analysis

In accord with the present invention, oligonucleotides were designed totarget DGAT2.

Shown in Table 19 is the sequence of each oligonucleotide. Plain textindicates a deoxynucleotide, and nucleotides designated with bold,underlined text are 2′-O-(2-methoxyethyl)nucleotides. Also shown foreach oligonucleotide in Table 19 is its motif, the target site on humanDGAT2 mRNA (GENBANK® accession number NM_(—)032564.2, incorporatedherein as SEQ ID NO: 110), and its cross-species identity. For eachspecies listed, an “X” denotes perfect complementarity to the targetsequence for that species, “1 MM” denotes one mismatch to the targetsequence for the species, etc.

TABLE 19 Antisense compounds targeting DGAT2 SEQ ID Target ISIS #Sequence NO Site Motif Human Monkey Rat Mouse 217328 GCATT GCCACTCCCATTCTT  95  909 5-10-5 X X  1 MM X 334177 AGGAC CCCGGAGTAG GCGGC  96  2465-10-5 X X  1 MM X 366710 GACCT ATTGAGCCAG GTGAC  97  396 5-10-5 X X 2 MM  2 MM 366714 GTAGC TGCTTTTCCA CCTTG  98  416 5-10-5 X X  2 MM 3 MM 370727 AG CTGCTTTTCCACCTTG GA  99  414 2-16-2 X X  2 MM  2 MM370747 TG GAGCTCAGAGACTCAG CC 100  953 2-16-2 X X  3 MM  2 MM 370784 GCTGCATCCATGTCATCA GC 101 2099 2-16-2 X X >3 MM >3 MM

Each of these oligonucleotides was tested in vitro for their ability toreduce human DGAT2 mRNA levels using real time RT-PCR methods asdescribed herein. In HepG2 and A549 cells, each of the oligonucleotidesin Table 19 demonstrated IC₅₀ values of about 20 nM.

Example 16 Effects of Gap-Widened Oligonucleotides on Reduction of DGAT2mRNA Levels—In Vivo Analysis

The oligonucleotides described in Table 19, along with ISIS 217357(ACACACTAGAAGTGAGCTTA, SEQ ID NO: 102), which is targeted to rat DGAT2,the complement of nucleotides 15333000 to 15365000 of GENBANK® accessionnumber NW_(—)047561.1, herein incorporated as SEQ ID NO: 111 were testedfor their ability to reduce DGAT2 levels in vivo. Eight week-old maleSprague-Dawley rats were injected with 20 mg/kg of oligonucleotide perweek for 2 weeks. Each treatment group was comprised of 6 animals.Animals injected with saline alone served as controls.

At the end of the treatment period, animals were sacrificed and liverand kidney tissues were harvested. To determine effects of altering thegap size on pharmacokinetics, oligonucleotide concentration in kidneyand liver were determined. Methods to determine oligonucleotideconcentration in tissues are known in the art (Geary et al., AnalBiochem, 1999, 274, 241-248). Total oligonucleotide is the sum of alloligonucleotides metabolites detected in the tissue. Shown in Table 20are the total concentration and the concentration of full lengtholigonucleotide (in μg/g) in the liver of animals treated with theindicated oligonucleotide concentration.

TABLE 20 Concentration of DGAT2 oligonucleotides in rat liver and kidneyTreatment Total Full length group Motif Liver Kidney Liver Kidney ISIS217357 5-10-5 91 441 70 328 ISIS 217328 5-10-5 145 399 121 294 ISIS334177 5-10-5 164 650 114 392 ISIS 366710 5-10-5 166 625 123 401 ISIS366714 5-10-5 278 674 214 488 ISIS 370727 2-16-2 209 355 131 166 ISIS370747 2-16-2 195 480 150 342 ISIS 370784 2-16-2 303 669 256 421

As shown in Table 20, kidney concentrations of gap-widenedoligonucleotides, particularly ISIS 370727 and ISIS 370747, weregenerally lower than those of oligonucleotides with a 10-deoxynucleotidegap.

Example 17 Effects of Gap-Widened Oligonucleotides on Reduction of DGAT2mRNA Levels—In Vivo Analysis

In another arm of the experiment described in Example 16, eight-week oldmale Sprague-Dawley rats were treated with the oligonucleotides at dosesof 50 mg/kg per week for four weeks. Each treatment group was comprisedof 4 animals. At the end of the treatment period, animals weresacrificed and target mRNA levels were determined using real-time RT-PCRas described herein. Results are shown in Table 21 as the average %inhibition for each treatment group.

TABLE 21 Reduction of target levels in rat liver with oligonucleotidestargeting DGAT2 Treatment % group Motif Inhibition ISIS 217357 5-10-5 25ISIS 217328 5-10-5 48 ISIS 334177 5-10-5 51 ISIS 366710 5-10-5 63 ISIS366714 5-10-5 67 ISIS 370727 2-16-2 77 ISIS 370747 2-16-2 79 ISIS 3707842-16-2 52

As shown in Table 21, the gap-widened oligonucleotides targeted to DGAT2show excellent inhibitory activity in the liver. ISIS 370727 and ISIS370747, in particular, showed superior ability to reduce targetexpression. Taken with the distribution of these oligonucleotides in theliver as shown in Table 20, these data suggest that gap-widenedoligonucleotides provide excellent to superior target reduction withoutenhanced accumulation of oligonucleotide in target tissues. In addition,the gap-widened oligonucleotides possess a preferred liver to kidneyratio as compared to the 5-10-5 motif oligonucleotides targeting DGAT2.

Example 18 Effects of Gap-Widened Oligonucleotides on Reduction of CRPmRNA Levels—In Vivo Analysis

Monkey-human cross-species oligonucleotides targeted to C-reactiveprotein (CRP) were designed to target CRP using sequences known in theart (see US application publication number US2005-0014257, hereinincorporated by reference in its entirety). Shown in Table 22 is thesequence of oligonucleotides targeted to CRP tested in cynomologusmonkeys. Plain text indicates a deoxynucleotide, and nucleotidesdesignated with bold, underlined text are2′-O-(2-methoxyethyl)nucleotides. Also shown for each oligonucleotide inTable 22 is its motif.

TABLE 22 Antisense oligonucleotides targeting CRP SEQ ID Isis # SequenceNO Motif 353512 TCC CATTTCAGGAGACC TGG 115 3-14-3 330012 TCCCATTTCAGGAGA CC TGG 115 5-10-5 353491 GCA CTCTGGACCCAAAC CAG 116 3-14-3133726 GCACT CTGGACCCAA ACCAG 116 5-10-5

Methods of assaying for activity of CRP compounds in vivo and in vitroare known in the art (see US application publication numberUS2005-0014257, herein incorporated by reference). Toxicity profiles ofgap-widened oligonucleotides were compared to the 5-10-5oligonucleotides by treating monkeys with 14 or 40 mg/kg/wk for 4 weeks.Activity was compared in a dose-escalation study with each cyclecontaining four subcutaneous doses administered (Mon., Wed., Fri., Mon.)in 4 dosing cycles over 8 weeks. Doses were 2, 4 and 10 mg/kg. At 48 hrfollowing the last dose in each treatment cycle, monkeys were challengedwith 1 to 2 μg/kg IL-6 (administered subcutaneously) and serum CRPlevels were quantified over 36 hours. Serum CRP levels may be measuredby ELISA using a commercially available kit (for example, ALerCHEK Inc.,Portland, Me.). Animals were sacrificed after the second and fourthcycles and liver CRP mRNA, tissue oligonucleotide concentration,clinical signs, serum chemistry, hematology, body weight, and histologywere assessed. With regard to tissue oligonucleotide concentration andhistology, the primary difference was 30% lower kidney concentration andfewer histologic changes in the 3-14-3 treated animals. Plasma cytokineand CRP levels were examined but not significantly increased.

Several CRP inhibitors were pharmacologically active, with the greatestreductions in serum CRP (30-66%) and hepatic CRP mRNA (60-85%) observedat both the 4 and 10 mg/kg treatment cycles.

We have surprisingly found that chimeric antisense compounds with gapsat least 11 nucleobases long and wings which are from independently from1 to 4 nucleobases in length which are 2′-MOE-modified. This enhancedefficacy is not predicted by the rank order potency of these compoundsin vitro (cell culture). 2-16-2 and 3-14-3 gapmer compounds as well as3-10-7 and 7-10-3 gapmer compounds have been shown to be more effectivethan 5-10-5 chimeras of the equivalent sequence and wing modification.4-12-4 gapmers are also believed to be a useful embodiment.

DETAILED DESCRIPTION OF EMBODIMENTS

Non-limiting examples of 2′-modified nucleosides useful in the compoundsof the present invention, include but are not limited to 2′-O-alkyl,2′-O-alkyl-O-alkyl wherein alkyl is a C₁ to C₆ alkyl or C₁ to C₆alkylene when alkyl is not a terminal substituent. These include2′-O-methyl, 2′-O-propyl and 2′-O-methoxyethyl nucleosides.

Details

The present invention uses antisense compounds which are chimericcompounds. “Chimeric” antisense compounds or “chimeras,” in the contextof this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide. These oligonucleotides typically contain atleast one region wherein the oligonucleotide is modified so as to conferupon the oligonucleotide increased resistance to nuclease degradation,increased cellular uptake, increased stability and/or increased bindingaffinity for the target nucleic acid. An additional region of theoligonucleotide may serve as a substrate for enzymes capable of cleavingRNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of oligonucleotide-mediatedinhibition of gene expression. The cleavage of RNA:RNA hybrids can, inlike fashion, be accomplished through the actions of endoribonucleases,such as RNAseL which cleaves both cellular and viral RNA. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed ascompositestructures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been, referred to in the artas hybrids or gapmers. Representative United States patents that teachthe preparation of such hybrid structures include, but are not limitedto, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; and 5,700,922, each of which is herein incorporated byreference in its entirety.

Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-ODimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidin-3′O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyldC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4-benzoyl5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N6benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N4isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl)nucleoside amidites,2′-(Dimethylaminooxyethoxy)nucleoside amidites,5′-O-tertButyldiphenylsilyl-O2-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,Ndimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy)nucleoside amidites,N2-isobutyryl6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives. Oligonucleotides:Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH4OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference. Alkyl phosphonate oligonucleotides are prepared as describedin U.S. Pat. No. 4,469,863, herein incorporated byreference.3′-Deoxy-3′-methylene phosphonate oligonucleotides areprepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference. Phosphoramidite oligonucleotides are preparedas described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878,herein incorporated by reference. Alkylphosphonothioate oligonucleotidesare prepared as described in published PCT applications PCT/US94/00902and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,respectively), herein incorporated by reference. 3′-Deoxy-3′-aminophosphoramidate oligonucleotides are prepared as described in U.S. Pat.No. 5,476,925, herein incorporated by reference. Phosphotriesteroligonucleotides are prepared as described in U.S. Pat. No. 5,023,243,herein incorporated by reference. Borano phosphate oligonucleotides areprepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, bothherein incorporated by reference. Oligonucleosides: Methylenemethyliminolinked oligonucleosides, also identified as MMI linked oligonucleosides,methylenedimethylhydrazo linked oligonucleosides, also identified as MDHlinked oligonucleosides, and methylenecarbonyl amino linkedoligonucleosides, also identified as amide-3 linked oligonucleosides,and methyleneaminocarbonyl linked oligonucleosides, also identified asamide-4 linked oligonucleosides, as well as mixed backbone compoundshaving, for instance, alternating MMI and P═O or P═S linkages areprepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677,5,602,240 and 5,610,289, all of which are herein incorporated byreference. Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference. Ethylene oxide linked oligonucleosides areprepared as described in U.S. Pat. No. 5,223,618, herein incorporated byreference.

RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized. RNAoligonucleotides are synthesized in a stepwise fashion. Each nucleotideis added sequentially (3′- to 5′-direction) to a solid support-boundoligonucleotide. The first nucleoside at the 3′-end of the chain iscovalently attached to a solid support. The nucleotide precursor, aribonucleoside phosphoramidite, and activator are added, coupling thesecond base onto the 5′-end of the first nucleoside. The support iswashed and any unreacted 5′-hydroxyl groups are capped with aceticanhydride to yield 5′-acetyl moieties. The linkage is then oxidized tothe more stable and ultimately desired P(V) linkage. At the end of thenucleotide addition cycle, the 5′-silyl group is cleaved with fluoride.The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S2Na2)in DMF. The deprotection solution is washed from the solid supportboundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55 ° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed,Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct. Additionally, methods of RNA synthesis are well known in theart (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996;Scaringe, S. A., et al., J. Am. Chem. Soc., 1″8, 120, 11820-11821;Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103,3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981,22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44,639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314;Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B.E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 tl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH4OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]; -[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′0-methyl chimeric oligonucleotide, with the substitutionof 2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O(methoxyethyl)phosphodiester]chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

The methods of the present invention are particularly useful inantisense therapeutics. It is not necessary that the antisense target beassociated with liver disease per se, since many current antisensetargets are expressed to high levels in liver and other organs. Inparticular, targets associated with metabolic and cardiovasculardiseases and conditions are particularly amenable to knockdown in theliver and have been shown in animals and in clinical studies to havetherapeutic effects).

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents.

1. A method of reducing expression of a target RNA in an animal, in needof reducing expression of said target RNA, comprising administering tosaid animal a gap-widened antisense oligonucleotide 18-24 linkednucleosides in length comprising: (a) a gap region having 12 to18contiguous 2′-deoxyribonucleosides; and (b) a first wing region having1 to 4 contiguous nucleosides; and (c) a second wing region having 1 to4 contiguous nucleosides; wherein the gap region is located between saidfirst wing region and said second wing region and, wherein eachnucleoside of said first and second wing region is a 2′modifiednucleoside thereby reducing expression of said target RNA in saidanimal. 2.-3. (canceled)
 4. The method of claim 1, wherein the targetRNA is associated with a metabolic or a cardiovascular disease orcondition.
 5. The method of claim 1, wherein the metabolic disease orcondition is selected from metabolic syndrome, diabetes, obesity,hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, Type 2diabetes, diet-induced obesity, hyperglycemia, insulin resistance,hepatic steatosis, fatty liver disease, or non-alcoholicsteatohepatitis.
 6. The method of claim 1, wherein the cardiovasculardisease or condition is selected from familial hypercholesterolemia,nonfamilial hypercholesterolemia, mixed dyslipidemia,dysbetalipoproteinemia, atherosclerosis, coronary artery disease,myocardial infarction, hypertension, carotid artery diseases, carotidartery disease, stroke, cerebrovascular disease, peripheral vasculardisease, thrombosis, or arterial aneurism.
 7. The method of claim 1,wherein the gap-widened antisense oligonucleotide has a wing-gap-wingmotif selected from 2-16-2, 3-14-3, 2-14-2, 3-12-3 or 4-12-4.
 8. Themethod of claim 1, wherein the gap-widened antisense oligonucleotide hasat least one phosphorothioate internucleotide linkage.
 9. The method ofclaim 6, wherein the gap-widened antisense oligonucleotide has allphosphorothioate internucleotide linkages.
 10. The method of claim 1,wherein gap-widened antisense oligonucleotide has at least one5-methylcytosine. 11.-25. (canceled)
 26. A method of modulating geneexpression in an animal comprising the step of contacting said animalwith the pharmaceutical composition comprising a gap-widened antisenseoligoncuelotide 18-24 linked nucleosides in length comprising: (a) a gapregion having 12 to 18 contiguous 2′-deoxyribonucleotides; (b) a firstwing region having 1 to 4 contiguous nucleosides; and (c) a second wingregion having 1 to 4 contiguous nucleosides; wherein the gap region islocated between said first wing region and said second wing region and,wherein each nucleoside of said first and second wing region is a 2′modified nucleoside thereby modulating gene expression in said animal.27.-28. (canceled)